Antibody-drug conjugate

ABSTRACT

As an antitumor drug which is excellent in terms of antitumor effect and safety, there is provided an antibody-drug conjugate in which an antitumor compound represented by the following formula is conjugated to an antibody via a linker having a structure represented by the following formula: -L1-L2-LF-NH—(CH2)n1-La-Lb-Lc- wherein the antibody is connected to the terminal of L1, and the antitumor compound is connected to the terminal of Lc with the nitrogen atom of the amino group at position 1 as a connecting position.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the Continuation of U.S. patent applicationSer. No. 16/142,354, filed on Sep. 26, 2018, which is a Continuation ofU.S. patent application Ser. No. 14/435,114, filed on Apr. 10, 2015(issued as U.S. Pat. No. 10,195,288 on Feb. 5, 2019), which is the U.S.National Phase of International Patent Application No.PCT/JP2013/006069, filed on Oct. 10, 2013, which claims priority toJapanese Application No. 2012-225887, filed on Oct. 11, 2012. The entirecontents of which are hereby incorporated by reference in theirentireties.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 20, 2018, isnamed sequence.txt and is 67 KB.

TECHNICAL FIELD

The present invention relates to an antibody-drug conjugate having anantitumor drug conjugated to an antibody capable of targeting tumorcells via a linker structure moiety, the conjugate being useful as anantitumor drug.

BACKGROUND ART

An antibody-drug conjugate (ADC) having a drug with cytotoxicityconjugated to an antibody, whose antigen is expressed on a surface ofcancer cells and which also binds to an antigen capable of cellularinternalization, and therefore can deliver the drug selectively tocancer cells and is thus expected to cause accumulation of the drugwithin cancer cells and to kill the cancer cells (see, Non PatentLiteratures 1 to 3). As an ADC, Mylotarg (Gemtuzumab ozogamicin) inwhich calicheamicin is conjugated to an anti-CD33 antibody is approvedas a therapeutic agent for acute myeloid leukemia. Further, Adcetris(Brentuximab vedotin), in which auristatin E is conjugated to ananti-CD30 antibody, has recently been approved as a therapeutic agentfor Hodgkin's lymphoma and anaplastic large cell lymphoma (see, NonPatent Literature 4). The drugs contained in ADCs which have beenapproved until now target DNA or tubulin.

With regard to an antitumor, low-molecular-weight compounds,camptothecin derivatives, compounds that inhibit topoisomerase I toexhibit an antitumor effect, are known. Among them, an antitumorcompound represented by the formula below

(exatecan, chemical name:(1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10,13(9H,15H)-dione)is a water soluble derivative of camptothecin (Patent Literature 1 and2). Unlike irinotecan currently used in clinical settings, an activationby an enzyme is unnecessary. Further, the inhibitory activity ontopoisomerase I is higher than SN-38 which is a main pharmaceuticallyactive substance of irinotecan and topotecan also used in clinicalsettings, and higher in vitro cytocidal activity is obtained for againstvarious cancer cells. In particular, it exhibits the effect againstcancer cells which have resistance to SN-38 or the like due toexpression of P-glycoprotein. Further, in a human tumor subcutaneouslytransplanted mouse model, it exhibited a potent antitumor effect, andthus has undergone the clinical studies, but has not been put on themarket yet (see, Non Patent Literatures 5 to 10). It remains unclearwhether or not exatecan functions effectively as an ADC.

DE-310 is a complex in which exatecan is conjugated to a biodegradablecarboxymethyldextran polyalcohol polymer via a GGFG peptide spacer(Patent Literature 3). By converting exatecan into a form of a polymerprodrug, so that a high blood retention property can be maintained andalso a high targetable property to a tumor area is passively increasedby utilizing the increased permeability of newly formed blood vesselswithin tumor and retention property in tumor tissues. With DE-310,through a cleavage of the peptide spacer by enzyme, exatecan andexatecan with glycine connected to an amino group are continuouslyreleased as a main active substance. As a result, the pharmacokineticsare improved and DE-310 was found to have higher effectiveness thanexatecan administered alone even though the dosage of exatecan is lowerthan the case of administration of exatecan alone according to varioustumor evaluation models in non-clinical studies. A clinical study wasconducted for DE-310, and effective cases were confirmed in humans, inwhich a report suggesting that the main active substance accumulates ina tumor than in normal tissues was present, however, there is also areport indicating that the accumulation of DE-310 and the main activesubstance in a tumor is not much different from the accumulation innormal tissues in humans, and thus no passive targeting is observed inhumans (see, Non Patent Literatures 11 to 14). As a result, DE-310 wasnot also commercialized, and it remains unclear whether or not exatecaneffectively functions as a drug oriented for such targeting.

As a compound relating to DE-310, a complex in which a structure moietyrepresented by —NH(CH₂)₄C(═O)— is inserted between -GGFG-spacer andexatecan to form -GGFG-NH(CH₂)₄C(═O)— used as a spacer structure is alsoknown (Patent Literature 4). However, the antitumor effect of thecomplex is not known at all.

CITATION LIST Patent Literature

-   [Patent Literature 1] Japanese Patent Laid-Open No. 5-59061-   [Patent Literature 2] Japanese Patent Laid-Open No. 8-337584-   [Patent Literature 3] International Publication No. WO 1997/46260-   [Patent Literature 4] International Publication No. WO 2000/25825

Non Patent Literature

-   [Non Patent Literature 1] Ducry, L., et al. Bioconjugate    Chem. (2010) 21, 5-13.; Antibody-Drug Conjugates: Linking cytotoxic    payloads to monoclonal antibodies.-   [Non Patent Literature 2] Alley, S. C., et al. Current Opinion in    Chemical Biology (2010) 14, 529-537.; Antibody-drug conjugates:    targeted drug delivery for cancer.-   [Non Patent Literature 3] Damle N. K. Expert Opin. Biol.    Ther. (2004) 4, 1445-1452.; Tumour-targeted chemotherapy with    immunoconjugates of calicheamicin.-   [Non Patent Literature 4] Senter P. D., et al. Nature    Biotechnology (2012) 30, 631-637.; The discovery and development of    brentuximab vedotin for use in relapsed Hodgkin lymphoma and    systemic anaplastic large cell lymphoma.-   [Non Patent Literature 5] Kumazawa, E., Tohgo, A., Exp. Opin.    Invest. Drugs (1998) 7, 625-632.; Antitumour activity of DX-8951f: a    new camptothecin derivative.-   [Non Patent Literature 6] Mitsui, I., Kumazawa, E., Hirota, Y., et    al. Jpn J. Cancer Res. (1995) 86, 776-782.; A new water-soluble    camptothecin derivative, DX-8951f, exhibits potent antitumor    activity against human tumors in vitro and in vivo.-   [Non Patent Literature 7] Takiguchi, S., Tohgo, A., et al. Jpn J.    Cancer Res. (1997) 88, 760-769.; Antitumor effect of DX-8951, a    novel camptothecin analog, on human pancreatic tumor cells and their    CPT-11-resistant variants cultured in vitro and xenografted into    nude mice.-   [Non Patent Literature 8] Joto, N. et al. Int J Cancer (1997) 72,    680-686.; DX-8951f, a water-soluble camptothecin analog, exhibits    potent antitumor activity against a human lung cancer cell line and    its SN-38-resistant variant.-   [Non Patent Literature 9] Kumazawa, E. et al. Cancer Chemother.    Pharmacol. (1998) 42, 210-220.; Potent and broad antitumor effects    of DX-8951f, a water-soluble camptothecin derivative, against    various human tumors xenografted in nude mice.-   [Non Patent Literature 10] De Jager, R., et al. Ann N Y Acad    Sci (2000) 922, 260-273.; DX-8951f: summary of phase I clinical    trials.-   [Non Patent Literature 11] Inoue, K. et al. Polymer Drugs in the    Clinical Stage, Edited by Maeda et al. (2003), 145-153.;    CM-dextran-polyalcohol-camptothecin conjugate, DE-310 with a novel    carrier system and its preclinical data.-   [Non Patent Literature 12] Kumazawa, E. et al. Cancer Sci (2004) 95,    168-175.; DE-310, a novel macromolecular carrier system for the    camptothecin analog DX-8951f: Potent antitumor activities in various    murine tumor models.-   [Non Patent Literature 13] Soepenberg, O. et al. Clinical Cancer    Research, (2005) 11, 703-711.; Phase I and pharmacokinetic study of    DE-310 in Patients with Advanced Solid Tumors.-   [Non Patent Literature 14] Wente M. N. et al. Investigational New    Drugs (2005) 23, 339-347.; DE-310, a macromolecular prodrug of the    topoisomerase-I-inhibitor exatecan (DX-8951), in patients with    operable solid tumors.

SUMMARY OF INVENTION Technical Problem

With regard to the treatment of tumor by an antibody, an insufficientantitumor effect may be observed even when the antibody recognizes anantigen and binds to tumor cells, and there is a case in which a moreeffective antitumor antibody is needed. Further, many antitumorlow-molecular-weight compounds have a problem in safety like side effectand toxicity even the compounds have an excellent antitumor effect, itremains as a subject to achieve a superior therapeutic effect by furtherenhancing the safety. Thus, an object of the present invention is toobtain to provide an antitumor drug having an excellent therapeuticeffect, which is excellent in terms of antitumor effect and safety.

Means to Solve the Problem

The inventors thought that, when an antitumor compound exatecan isconverted into an antibody-drug conjugate, via a linker structuremoiety, by conjugation to the antibody, which is capable of targetingtumor cells, that is having a property of recognizing tumor cells, aproperty of binding to tumor cells, a property of internalizing withintumor cells, a cytocidal activity against tumor cells, or the like, theantitumor compound can be more surely delivered to tumor cells tospecifically exhibit the antitumor effect of the compound in tumorcells, and thus the antitumor effect can be surely exhibited and also anenhanced cytocidal effect of the antibody is expected, and a dose of theantitumor compound can be reduced compared to a case of administeringthe compound alone, and thus an influence of the antitumor compound onnormal cells can be alleviated so that higher safety can be achieved.

In this connection, the inventors created a linker with a specificstructure and succeeded in obtaining an antibody-drug conjugate in whichthe antibody and exatecan are conjugated to each other via the linker,and confirmed an excellent antitumor effect exhibited by the conjugateto thereby complete the present invention.

Specifically, the present invention relates to the followings.

[1] An antibody-drug conjugate wherein an antitumor compound representedby the following formula:

is conjugated to an antibody via a linker having a structure representedby the following formula:

-L¹-L²-L^(P)-NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)-.

Here, the antibody is connected to the terminal of L¹, the antitumorcompound is connected to the terminal of L^(c) with the nitrogen atom ofthe amino group at position 1 as connecting position, wherein

n¹ represents an integer of 0 to 6,L¹ represents -(Succinimid-3-yl-N)—(CH₂)n²-C(═O)—,—CH₂—C(═O)—NH—(CH₂)n³-C(═O)—,—C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)-, or —C(═O)—(CH₂)n⁴-C(═O)—,

wherein n² represents an integer of 2 to 8, n³ represents an integer of1 to 8, n⁴ represents an integer of 1 to 8,

L² represents —NH—(CH₂—CH₂-0)n⁵-CH₂—CH₂—C(═O)—, —S—(CH₂)n⁶-C(═O)—, or asingle bond, wherein n⁵ represents an integer of 1 to 6, n⁶ representsan integer of 1 to 6,L^(P) represents a peptide residue consisting of 2 to 7 amino acids,L^(a) represents —C(═O)—NH—, —NR¹—(CH₂)n⁷-, —O—, or a single bond,

wherein n⁷ represents an integer of 1 to 6, R¹ represents a hydrogenatom, an alkyl group having 1 to carbon atoms, —(CH₂)n⁸-COOH, or—(CH₂)n⁹-OH, n⁸ represents an integer of 1 to 4, n⁹ represents aninteger of 1 to 6,

L^(b) represents —CR²(—R³)—, —O—, —NR⁴—, or a single bond,

wherein R² and R³ each independently represents a hydrogen atom, analkyl group having 1 to 6 carbon atoms, —(CH₂)n^(a)-NH₂,—(CH₂)n^(b)-COOH, or —(CH₂)n^(c)-OH, R⁴ represents a hydrogen atom or analkyl group having 1 to 6 carbon atoms, n^(a) represents an integer of 0to 6, n^(b) represents an integer of 1 to 4, n^(c) represents an integerof 1 to 4, provided that when n^(a) is 0, R² and R³ are not the same aseach other,

L^(c) represents —CH₂— or —C(═O)—,-(Succinimid-3-yl-N)— has a structure represented by the followingformula:

which is connected to the antibody at position 3 thereof and isconnected to a methylene group in the linker structure containing thisstructure on the nitrogen atom at position 1,—(N-ly-3-diminiccuS)- has a structure represented by the followingformula:

which is connected to L² at position 3 thereof and is connected to amethylene group in the linker structure containing this structure on thenitrogen atom at position 1,cyc.Hex(1,4) represents a 1,4-cyclohexylene group, and when L² is—S—(CH₂)n⁶-C(═O)—, L¹ is —C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)-.

The present invention further relates to each of the followings.

[2] The antibody-drug conjugate according to [1], wherein L^(c) is—C(═O)—.[3] The antibody-drug conjugate according to [1] or [2], wherein thebond between the antibody and L¹ is a thioether bond which is formed ata disulfide bond site present in a hinge part of the antibody,a disulfide bond which is formed at a disulfide bond site present in ahinge part of the antibody, oran amide bond which is formed at an amino group present on a side chainof an amino acid constituting the antibody or at the terminal aminogroup.[4] The antibody-drug conjugate according to any one of [1] to [3],wherein the peptide residue of L^(P) is an amino acid residue comprisingan amino acid selected from phenylalanine, glycine, valine, lysine,citrulline, serine, glutamic acid, and aspartic acid.[5] The antibody-drug conjugate according to any one of [1] to [3],wherein L^(P) is a peptide residue consisting of 4 amino acids.[6] The antibody-drug conjugate according to any one of [1] to [3],wherein L^(P) is -GGFG-.[7] An antibody-drug conjugate wherein an antitumor compound representedby the following formula:

is conjugated to an antibody via a linker having a structure representedby the following formula:

L¹-L²-L^(P)-NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)-.

Here, the antibody is connected to the terminal of L¹, the antitumorcompound is connected to the terminal of L^(c) with the nitrogen atom ofthe amino group at position 1 as a connecting position,

whereinn¹ represents an integer of 0 to 6,L¹ represents -(Succinimid-3-yl-N)—(CH₂)n²-C(═O)—,—CH₂—C(═O)—NH—(CH₂)n²-C(═O)—,—C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)-, or —C(═O)—(CH₂)n⁴-C(═O)—,

wherein n² represents an integer of 2 to 8, n³ represents an integer of1 to 8, n⁴ represents an integer of 1 to 8,

L² represents —NH—(CH₂—CH₂-0)n⁵-CH₂—CH₂—C(═O)—, —S—(CH₂)n⁶-C(═O)—, or asingle bond,

wherein n⁵ represents an integer of 1 to 6, n⁶ represents an integer of1 to 6,

L^(P) represents a tetrapeptide residue of GGFG,L^(a) represents —O— or a single bond,L^(b) represents —CR²(—R²)— or a single bond,

wherein R² and R³ each represents a hydrogen atom, L^(c) represents—C(═O)—,

-(Succinimid-3-yl-N)— has a structure represented by the followingformula:

which is connected to the antibody at position 3 thereof and isconnected to a methylene group in the linker structure containing thisstructure on the nitrogen atom at position 1,—(N-ly-3-diminiccuS)- has a structure represented by the followingformula:

which is connected to L² at position 3 thereof and is connected to amethylene group in the linker structure containing this structure on thenitrogen atom at position 1,cyc.Hex(1,4) represents a 1,4-cyclohexylene group, and when L² is—S—(CH₂)n⁶-C(═O)—, L¹ is —C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)-.[8] The antibody-drug conjugate according to any one of [1] to [7],wherein L¹ is -(Succinimid-3-yl-N)—(CH₂)n²-C(═O)— or—CH₂—C(═O)—NH—(CH₂)n³-C(═O)—.[9] The antibody-drug conjugate according to any one of [1] to [7],wherein L¹ is -(Succinimid-3-yl-N)—(CH₂)n²-C(═O)—.[10] The antibody-drug conjugate according to any one of [1] to [7],wherein L¹ is —C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)- or—C(═O)—(CH₂)n⁴-C(═O)—.[11] The antibody-drug conjugate according to any one of [1] to [9],wherein n² is an integer of 2 to 5, and L² is a single bond.[12] The antibody-drug conjugate according to any one of [1] to [9],wherein n² is an integer of 2 to 5, L² is—NH—(CH₂CH₂O)n⁵-CH₂—CH₂—C(═O)—, and n⁵ is 2 or 4.[13] The antibody-drug conjugate according to any one of [1] to [12],wherein —NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)- is a partial structure having achain length of 4 to 7 atoms.[14] The antibody-drug conjugate according to any one of [1] to [12],wherein —NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)- is a partial structure having achain length of 5 or 6 atoms.[15] The antibody-drug conjugate according to any one of [1] to [14],wherein —NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)- is—NH—(CH₂)₂—C(═O)—,—NH—CH₂—O—CH₂—C(═O)—, or—NH—(CH₂)₂—O—CH₂—C(═O)—.[16] The antibody-drug conjugate according to any one of [1] to [15],wherein the drug-linker structure moiety is one drug-linker structureselected from the group consisting of the following drug-linkerstructures:-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)

-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)    —C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)    —C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)—S—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)

Wherein, -(Succinimid-3-yl-N)— has a structure represented by thefollowing formula:

which is connected to the antibody at position 3 thereof and isconnected to a methylene group in the linker structure containing thisstructure on the nitrogen atom at position 1,—(N-ly-3-diminiccuS)- has a structure represented by the followingformula:

which is connected to L² at position 3 thereof and is connected to amethylene group in the linker structure containing this structure on thenitrogen atom at position 1,cyc.Hex(1,4) represents a 1,4-cyclohexylene group,—(NE-DX) represents a group represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is theconnecting position, and-GGFG- represents a peptide residue of -Gly-Gly-Phe-Gly-.[17] The antibody-drug conjugate according to any one of [1] to [9] and[11] to [14], wherein the drug-linker structure moiety having a drugconnected to -L¹-L²-L^(P)-NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)- is onedrug-linker structure selected from the following group:-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX).

In the above, -(Succinimid-3-yl-N)— has a structure represented by thefollowing formula:

which is connected to the antibody at position 3 thereof and connectedto a methylene group in the linker structure containing this structureon the nitrogen atom at position 1, and—(NH-DX) represents a group represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is aconnecting position.[18] An antibody-drug conjugate wherein an antitumor compoundrepresented by the following formula:

is conjugated to an antibody via a linker having a structure representedby the following formula:

-L¹-L²-L^(P)-NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)-.

Here, the antibody is connected to the terminal of L¹, the antitumorcompound is connected to the terminal of L^(c),

whereinn¹ represents an integer of 0 to 6,L¹ represents -(Succinimid-3-yl-N)—(CH₂)n²-C(═O)— and is connected tothe antibody via a thioether bond which is formed at a disulfide bondsite present in a hinge part of the antibody,

wherein n² represents an integer of 2 to 8,

L² represents —NH—(CH₂—CH₂-0)n⁵-CH₂—CH₂—C(═O)— or a single bond,

wherein n⁵ represents an integer of 1 to 6,

L^(P) represents a tetrapeptide residue of GGFG,L^(a) represents —O— or a single bond,L^(b) represents —CR²(—R³)— or a single bond,

wherein R² and R³ each represents a hydrogen atom,

L^(c) represents —C(═O)—, and-(Succinimid-3-yl-N)— has a structure represented by the followingformula:

which is connected to the antibody at position 3 thereof and isconnected to a methylene group in the linker structure containing thisstructure on the nitrogen atom at position 1.[19] The antibody-drug conjugate according to [18], whereinn² is 2, L² is —NH—(CH₂—CH₂—O)n⁵-CH₂—CH₂—C(═O)—, n⁵ is 2,n¹ is 3, and both of L^(a) and L^(b) are single bonds,n² is 5, L² is a single bond, n¹ is 1, L^(a) is —O—, and L^(b) is—CR²(—R³)—, orn² is 5, L² is a single bond, n¹ is 2, L^(a) is —O—, and L^(b) is —CR²(—R³)—.[20] The antibody-drug conjugate according to [18] or [19], wherein n²is an integer of 2 to 5, and L² is a single bond.[21] The antibody-drug conjugate according to [18] or [19], wherein n²is an integer of 2 to 5, L² is —NH—(CH₂CH₂O) n⁵-CH₂—CH₂—C(═O)—, and n⁵is 2 or 4.[22] The antibody-drug conjugate according to any one of [18] to [21],wherein —NH—(CH₂) n¹-L^(a)-L^(b)-L^(c)- is

-   —NH—(CH₂)₃—C(═O)—,-   —NH—CH₂—O—CH₂—C(═O)—, or-   —NH—(CH₂)₂—O—CH₂—C(═O)—.    [23] The antibody-drug conjugate according to any one of [18] to    [22], wherein the drug-linker structure moiety is one drug-linker    structure selected from the group consisting of the following    drug-linker structures:-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)

In the above, -(Succinimid-3-yl-N)— has a structure represented by thefollowing formula:

which is connected to the antibody at position 3 thereof and isconnected to a methylene group in the linker structure containing thisstructure on the nitrogen atom at position 1, and—(NH-DX) represents a group represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is aconnection position.[24] The antibody-drug conjugate according to [23], wherein thedrug-linker structure moiety having a drug connected to-L¹-L²-L^(P)-NH—(CH₂)n¹-L^(a)-L^(b)-L^(e)- is one drug-linker structureselected from the following group:

-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX).

In the above, -(Succinimid-3-yl-N)— has a structure represented by thefollowing formula:

which is connected to the antibody at position 3 thereof and isconnected to a methylene group in the linker structure containing thisstructure on the nitrogen atom at position 1, and—(NH-DX) represents a group represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is aconnecting position.[25] The antibody-drug conjugate according to any one of [1] to [24],wherein an average number of units of the selected one drug-linkerstructure conjugated per antibody is in a range of from 1 to 10.[26] The antibody-drug conjugate according to any one of [1] to [24],wherein an average number of units of the selected one drug-linkerstructure conjugated per antibody is in a range of from 2 to 8.[27] The antibody-drug conjugate according to any one of [1] to [24],wherein an average number of units of the selected one drug-linkerstructure conjugated per antibody is in a range of from 3 to 8.[28] The antibody-drug conjugate according to any one of [1] to [27],wherein the antibody is an antibody having one or more of a property ofrecognizing a target cell, a property of binding to a target cell, aproperty of internalizing in a target cell, and a property of damaging atarget cell.[29] The antibody-drug conjugate according to any one of [1] to [27],wherein a cell which is targeted by the antibody-drug conjugate is atumor cell.[30] The antibody-drug conjugate according to any one of [1] to [27],wherein the antibody is an anti-A33 antibody, an anti-B7-H3 antibody, ananti-CanAg antibody, an anti-CD20 antibody, an anti-CD22 antibody, ananti-CD30 antibody, an anti-CD33 antibody, an anti-CD56 antibody, ananti-CD70 antibody, an anti-CEA antibody, an anti-Cripto antibody, ananti-EphA2 antibody, an anti-G250 antibody, an anti-MUC1 antibody, ananti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody,an anti-tenascin-C antibody, an anti-SLC44A4 antibody, or ananti-mesothelin antibody.[31] The antibody-drug conjugate according to any one of [1] to [27],wherein the antibody is an anti-B7-H3 antibody, an anti-CD30 antibody,an anti-CD33 antibody, or an anti-CD70 antibody.[32] The antibody-drug conjugate according to any one of [1] to [27],wherein the antibody is an anti-B7-H3 antibody.[33] A drug containing the antibody-drug conjugate according to any oneof [1] to [32], a salt thereof or a hydrate thereof.[34] An antitumor drug and/or anticancer drug containing theantibody-drug conjugate according to any one of [1] to [32], a saltthereof or a hydrate thereof.[35] The antitumor drug and/or anticancer drug according to [34], whichis applied to lung cancer, kidney cancer, urothelial cancer, colorectalcancer, prostate cancer, glioblastoma multiforme, ovarian cancer,pancreatic cancer, breast cancer, melanoma, liver cancer, bladdercancer, stomach cancer, or esophageal cancer.[36] A pharmaceutical composition containing the antibody-drug conjugateaccording to any one of [1] to [32], a salt thereof or a hydrate thereofas an active component, and a pharmaceutically acceptable formulationcomponent.[37] The pharmaceutical composition according to [36], which is appliedto lung cancer, kidney cancer, urothelial cancer, colorectal cancer,prostate cancer, glioblastoma multiforme, ovarian cancer, pancreaticcancer, breast cancer, melanoma, liver cancer, bladder cancer, stomachcancer, or esophageal cancer.[38] A method for treating tumor and/or cancer comprising administeringthe antibody-drug conjugate according to any one of [1] to [32], a saltthereof or a hydrate thereof.[39] A drug-linker intermediate compound represented by the followingformula:

Q-(CH₂)n^(Q)-C(═O)-L^(2a)-L^(P)-NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)-(NH-DX).

In the formula, Q represents (maleimid-N-yl)-, HS—, X—CH₂—C(═O)—NH—, or(Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—,

X represents a bromine atom or an iodine atom,n⁴ represents an integer of 2 to 8,L^(2a) represents —NH—(CH₂—CH₂—O)n⁵-CH₂—CH₂—C(═O)— or a single bond,

wherein n⁵ represents an integer of 1 to 6,

L^(P) represents a peptide residue consisting of 2 to 7 amino acidsselected from phenylalanine, glycine, valine, lysine, citrulline,serine, glutamic acid, and aspartic acid,n¹ represents an integer of 0 to 6,L^(a) represents —C(═O)—NH—, —NR¹—(CH₂)n⁷-, —O—, or a single bond,

wherein n⁷ represents an integer of 1 to 6, R¹ represents a hydrogenatom, an alkyl group having 1 to carbon atoms, —(CH₂)n⁸-COOH, or—(CH₂)n⁹-OH, n⁸ represents an integer of 1 to 4, n⁹ represents aninteger of 1 to 6,

L^(b) represents —CR²(—R³)—, —O—, —NR⁴—, or a single bond,

wherein R² and R³ each independently represent a hydrogen atom, an alkylgroup having 1 to 6 carbon atoms, —(CH₂)n^(a)-NH₂, —(CH₂)n^(b)-COOH, or—(CH₂)n^(c)-OH, R⁴ represents a hydrogen atom or an alkyl group having 1to 6 carbon atoms, n^(a) represents an integer of 0 to 6, n^(b)represents an integer of 1 to 4, n^(c) represents an integer of 1 to 4,provided that when n^(a) is 0, R² and R³ are not the same as each other,

L^(c) represents —CH₂— or —C(═O)—,(maleimid-N-yl)- is a group represented by the following formula:

wherein the nitrogen atom is a connecting position,(Pyrrolidine-2,5-dione-N-yl) is a group represented by the followingformula:

wherein the nitrogen atom is a connecting position, and —(NH-DX) is agroup represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is aconnecting position.[40] The drug-linker intermediate compound according to [39], whereinL^(c) is —C(═O)—.[41] The drug-linker intermediate compound according to [39] or [40],wherein L^(P) is a peptide residue consisting of 4 amino acids.[42] The drug-linker intermediate compound according to any one of [39]to [41], wherein L^(P) is -GGFG-.[43] The drug-linker intermediate compound according to any one of [39]to [42], wherein —NH—(CH₂)n¹-L^(a)-L^(b)- is—NH—CH₂CH₂—,—NH—CH₂CH₂CH₂—,—NH—CH₂CH₂CH₂CH₂—,—NH—CH₂CH₂CH₂CH₂CH₂—,—NH—CH₂—O—CH₂—, or—NH—CH₂CH₂—O—CH₂—.[44] The drug-linker intermediate compound according to any one of [39]to [42], wherein —NH—(CH₂)n¹-L^(a)-L^(b)- is —NH—CH₂CH₂CH₂—,—NH—CH₂—O—CH₂—, or—NH—(CH₂)₂—O—CH₂—.[45] The drug-linker intermediate compound according to any one of [39]to [44], wherein n^(Q) is an integer of 2 to 6.[46] The drug-linker intermediate compound according to [43], whereinQ is (maleimid-N-yl)-,n^(Q) is an integer of 2 to 5, andL^(2a) is a single bond.[47] The drug-linker intermediate compound according to [44], whereinQ is (maleimid-N-yl)-,n^(Q) is an integer of 2 to 5, andL^(2a) is a single bond.[47] The drug-linker intermediate compound according to any one of [39]to [42], whereinQ is (maleimid-N-yl)-,n^(Q) is an integer of 2 to 5,L^(2a) is —NH—(CH₂—CH₂—O)n⁵-CH₂—CH₂—C(═O)—,n⁵ is an integer of 2 to 4, and—NH—(CH₂)n¹-L^(a)-L^(b)- is—NH—CH₂CH₂—,—NH—CH₂CH₂CH₂—,—NH—CH₂CH₂CH₂CH₂—,—NH—CH₂CH₂CH₂CH₂CH₂—,—NH—CH₂—O—CH₂—, or—NH—CH₂CH₂—O—CH₂—.[49] The drug-linker intermediate compound according to [48], whereinn⁵ is an integer of 2 or 4, and—NH—(CH₂)n¹-L^(a)-L^(b)- is—NH—CH₂CH₂CH₂—,—NH—CH₂—O—CH₂—, or—NH—CH₂CH₂—O—CH₂—.[50] A compound of the following:

-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—    CH₂— C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—    C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   X— CH₂— C(═O)—NH—CH₂CH₂— C(═O)-GGFG-NH—CH₂CH₂—O— CH₂— C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂— C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)    X— CH₂— C(═O)—NH—CH₂CH₂CH₂CH₂CH₂—    C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   X— CH₂— C(═O)—NH—CH₂CH₂— C(═O)—NH—CH₂CH₂O—CH₂CH₂O—    CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   X— CH₂— C(═O)—NH—CH₂CH₂— C(═O)—NH—CH₂CH₂O—CH₂CH₂O—    CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—    C(═O)—NH—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—    C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂— C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂— C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O— CH₂— C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂— C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂— C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂— C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂— C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—    C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—    C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—    C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—    C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—    C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—    C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)    (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂-0-CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),    or-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX).

In the above, (maleimid-N-yl)- is a group represented by the followingformula:

wherein the nitrogen atom is a connecting position, X represents ahalogen atom,

-   (Pyrrolidine-2,5-dione-N-yl)- is a group represented by the    following formula:

wherein the nitrogen atom is a connecting position, and —(NH-DX) is agroup represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is aconnecting position.

A compound of the following:

-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—    C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),    or-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—    C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX).

In the above, (maleimid-N-yl)- is a group represented by the followingformula:

wherein the nitrogen atom is a connecting position, and —(NH-DX) is agroup represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is aconnecting position.[52] A compound of the following:

-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)    or-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX).

In the above, (maleimid-N-yl)- is a group represented by the followingformula:

wherein the nitrogen atom is a connecting position, and —(NH-DX) is agroup represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is aconnecting position.

A compound selected from the following group:

NH₂—CH₂CH₂—C(═O)—(NH-DX),NH₂—CH₂CH₂CH₂—C(═O)—(NH-DX),NH₂—CH₂—O—CH₂—C(═O)—(NH-DX),NH₂—CHCH₂—O—CH₂—C(═O)—(NH-DX), and

HO—CH₂—C(═O)—(NH-DX)

wherein —(NH-DX) is a group represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is aconnecting position.[54] A compound represented by the following formula:

[55] A compound represented by the following formula:

[56] A compound represented by the following formula:

A method for producing an antibody-drug conjugate comprising reacting acompound represented by the following formula:

Q-(CH₂)n^(Q)-C(═O)-L^(2a)-L^(P)-NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)-(NH-DX)

with an antibody or a reactive derivative thereof and conjugating adrug-linker moiety to the antibody by a method for forming a thioetherbond at a disulfide bond site present in a hinge part of the antibody,or by a method for forming an amide bond at an amino group present on aside chain of an amino acid constituting the antibody or at the terminalamino group.

In the formula, Q represents (maleimid-N-yl)-, HS—, X—CH₂—C(═O)—NH—, or(Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—,

X represents a bromine atom or an iodine atom,n^(Q) represents an integer of 2 to 8,L^(2a) represents —NH—(CH₂—CH₂—O)n⁵-CH₂—CH₂—C(═O)— or a single bond,

wherein n⁵ represents an integer of 1 to 6,

L^(P) represents a peptide residue consisting of 2 to 7 amino acidsselected from phenylalanine, glycine, valine, lysine, citrulline,serine, glutamic acid, and aspartic acid,n¹ represents an integer of 0 to 6,L^(a) represents —C(═O)—NH—, —NR′—(CH₂)n⁷-, —O—, or a single bond,

wherein n⁷ represents an integer of 1 to 6, R¹ represents a hydrogenatom, an alkyl group having 1 to carbon atoms, —(CH₂) n⁸-COOH, or—(CH₂)n⁹-OH, n⁸ represents an integer of 1 to 4, n⁹ represents aninteger of 1 to 6,

L^(b) represents —CR²(—R³)—, —O—, —NR⁴—, or a single bond,

wherein R² and R³ each independently represents a hydrogen atom, analkyl group having 1 to 6 carbon atoms, —(CH₂) n^(a)-NH₂, —(CH₂)n^(b)-COOH, or —(CH₂)n^(c)-OH, R⁴ represents a hydrogen atom or an alkylgroup having 1 to 6 carbon atoms, n^(a) represents an integer of 0 to 6,n^(b) represents an integer of 1 to 4, n^(c) represents an integer of 1to 4, provided that when n′ is 0, R² and R³ are not the same as eachother,

L^(c) represents —CH₂— or —C(═O)—,(maleimid-N-yl)- is a group represented by the following formula:

wherein the nitrogen atom is a connecting position,(Pyrrolidine-2,5-dione-N-yl) is a group represented by the followingformula:

wherein the nitrogen atom is a connecting position, and —(NH-DX) is agroup represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is aconnecting position.[58] The production method according to [57], wherein the method forconjugating a drug-linker moiety to an antibody isa method of reducing the antibody and thereafter forming a thioetherbond by the reaction with the compound in which Q is a maleimidyl groupor X—CH₂—C(═O)—NH—,a method of forming an amide bond by the reaction with the compound inwhich Q is (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—, ora method of reacting the antibody with a compound represented by theformula Q¹-L^(1a)-Q²[wherein Q¹ represents (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—,(3-Sulfo-pyrrolidine-2,5-dione-N-yl)-O—C(═O)—, R^(Q)—O—C(═N)—, orO═C═N—,L^(1a)- represents -cyc.Hex(1,4)-CH₂—, an alkylene group having 1 to 10carbon atoms, a phenylene group, —(CH₂)n⁴-C(═O)—, —(CH₂)n^(4a)-NH—C(═O)—(CH₂)n^(4b-), or—(CH₂)n^(4a)-NH—C(═O)-cyc.Hex(1,4)-CH₂—,Q² represents (maleimid-N-yl), a halogen atom, or —S—S-(2-Pyridyl),R^(Q) represents an alkyl group having 1 to 6 carbon atoms,n⁴ represents an integer of 1 to 8,n^(4a) represents an integer of 0 to 6, n^(4b) represents an integer of1 to 6,(3-Sulfo-pyrrolidine-2,5-dione-N-yl)- is a group represented by thefollowing formula:

wherein the nitrogen atom is a connecting position, and this sulfonicacid is capable of forming a lithium salt, sodium salt, or potassiumsalt,cyc.Hex(1,4) represents a 1,4-cyclohexylene group, and (2-Pyridyl)represents a 2-pyridyl group]and thereafter reacting with the compound in which Q is SH to form adrug-linker structure by an amide bond.[59] The production method according to [57] or [58], wherein an averagenumber of units of the selected one drug-linker structure conjugated perantibody is in a range of from 1 to 10.[60] The production method according to [57] or [58], wherein an averagenumber of units of the selected one drug-linker structure conjugated perantibody is in a range of from 2 to 8.[61] The production method according to [57] or [58], wherein an averagenumber of units of the selected one drug-linker structure conjugated perantibody is in a range of from 3 to 8.[62] The production method according to any one of [57] to [61], whereina cell which is targeted by the antibody-drug conjugate is a tumor cell.[63] The production method according to any one of [57] to [61a],wherein the antibody is an anti-A33 antibody, an anti-B7-H3 antibody, ananti-CanAg antibody, an anti-CD20 antibody, an anti-CD22 antibody, ananti-CD30 antibody, an anti-CD33 antibody, an anti-CD56 antibody, ananti-CD70 antibody, an anti-CEA antibody, an anti-Cripto antibody, ananti-EphA2 antibody, an anti-G250 antibody, an anti-MUC1 antibody, ananti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody,an anti-tenascin-C antibody, an anti-SLC44A4 antibody, or ananti-mesothelin antibody.[64] The production method according to any one of [57] to [61], whereinthe antibody is an anti-B7-H3 antibody, an anti-CD30 antibody, ananti-CD33 antibody, or an anti-CD70 antibody.[65] The production method according to any one of [57] to [61], whereinthe antibody is an anti-B7-H3 antibody.[66] An antibody-drug conjugate obtained by the production methodaccording to any of [57] to [65].[67] An antibody-drug conjugate obtained by forming a thioether bond ata sulfide bond site in a hinge part of an antibody, wherein the antibodyis treated in a reducing condition and thereafter reacted with acompound selected from the compound group shown below:

-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),    or-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX).

In the above, (maleimid-N-yl)- is a group represented by the followingformula:

wherein the nitrogen atom is a connecting position, and —(NH-DX) is agroup represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is aconnecting position.[68] An antibody-drug conjugate obtained by forming a thioether bond ata sulfide bond site present in a hinge part of an antibody, wherein theantibody is treated in a reducing condition and thereafter reacted witha compound selected from the compound group shown below:

-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),    or-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX).

In the above, (maleimid-N-yl)- is a group represented by the followingformula:

wherein the nitrogen atom is a connecting position, and —(NH-DX) is agroup represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is aconnecting position.[69] The antibody-drug conjugate according to [67] or [68], wherein anaverage number of units of the selected one drug-linker structureconjugated per antibody is in a range of from 1 to 10.[70] The antibody-drug conjugate according to [67] or [68], wherein anaverage number of units of the selected one drug-linker structureconjugated per antibody is in a range of from 2 to 8.[71] The antibody-drug conjugate according to [67] or [68], wherein anaverage number of units of the selected one drug-linker structureconjugated per antibody is in a range of from 3 to 8.[72] The antibody-drug conjugate according to any one of [67] to [71],wherein a cell which is targeted by the antibody-drug conjugate is atumor cell.[73] The antibody-drug conjugate according to any one of [67] to [71],wherein the antibody is an anti-A33 antibody, an anti-B7-H3 antibody, ananti-CanAg antibody, an anti-CD20 antibody, an anti-CD22 antibody, ananti-CD30 antibody, an anti-CD33 antibody, an anti-CD56 antibody, ananti-CD70 antibody, an anti-CEA antibody, an anti-Cripto antibody, ananti-EphA2 antibody, an anti-G250 antibody, an anti-MUC1 antibody, ananti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody,an anti-tenascin-C antibody, an anti-SLC44A4 antibody, or ananti-mesothelin antibody.[74] The antibody-drug conjugate according to any one of [67] to [71],wherein the antibody is an anti-B7-H3 antibody, an anti-CD30 antibody,an anti-CD33 antibody, or an anti-CD70 antibody.[75] The antibody-drug conjugate according to any one of [67] to [71],wherein the antibody is an anti-B7-H3 antibody.[76] A linker represented by the following formula:

-L¹-L²-L^(P)-NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)-

for obtaining an antibody-drug conjugate in which a drug is conjugatedto an antibody via the linker.

In the above, L¹ is a connecting position for the antibody, L^(c) is aconnecting position for an antitumor compound,

whereinn¹ represents an integer of 0 to 6,L¹ represents -(Succinimid-3-yl-N)—(CH₂)n²-C(═O)—,—CH₂—C(═O)—NH—(CH₂)n³-C(═O)—,—C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)-, or —C(═O)—(CH₂)n⁴-C(═O)—,

wherein n² represents an integer of 2 to 8, n³ represents an integer of1 to 8, n⁴ represents an integer of 1 to 8,

L² represents —NH—(CH₂—CH₂—O)n⁵-CH₂—CH₂—C(═O)—, —S—(CH₂)n⁶-C(═O)—, or asingle bond,

wherein n⁵ represents an integer of 1 to 6, n⁶ represents an integer of1 to 6,

L^(P) represents a peptide residue consisting of 2 to 7 amino acids,L^(a) represents —C(═O)—NH—, —NR¹—(CH₂)n⁷-, —O—, or a single bond,

wherein n⁷ represents an integer of 1 to 6, R¹ represents a hydrogenatom, an alkyl group having 1 to carbon atoms, —(CH₂)n⁸-COOH, or—(CH₂)n⁹-OH, n⁸ represents an integer of 1 to 4, n⁹ represents aninteger of 1 to 6,

L^(b) represents —CR²(—R³)—, —O—, —NR⁴—, or a single bond,

wherein R² and R³ each independently represents a hydrogen atom, analkyl group having 1 to 6 carbon atoms, —(CH₂) n^(a)-NH₂, —(CH₂)n^(b)-COOH, or —(CH₂)n^(c)-OH, R⁴ represents a hydrogen atom or an alkylgroup having 1 to 6 carbon atoms, n^(a) represents an integer of 0 to 6,n^(b) represents an integer of 1 to 4, n^(c) represents an integer of 1to 4, provided that when n^(a) is 0, R² and R³ are not the same eachother,

L^(c) represents —CH₂— or —C(═O)—,-(Succinimid-3-yl-N)— has a structure represented by the followingformula:

which is connected to the antibody at position 3 thereof and isconnected to a methylene group in the linker structure containing thisstructure on the nitrogen atom at position 1,—(N-ly-3-diminiccuS)- has a structure represented by the followingformula:

which is connected to L² at position 3 thereof and is connected to amethylene group in the linker structure containing this structure on thenitrogen atom at position 1,cyc.Hex(1,4) represents a 1,4-cyclohexylene group, and when L² is—S—(CH₂)n⁶-C(═O)—, L¹ is —C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)-.[77] The linker according to [76], which is selected from the followinggroup, provided that the left terminal is a connecting position with theantibody and the right terminal is a connecting position with theantitumor compound:

-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—    C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—    C(═O)-GGFG-NH—CH₂CH₂— C(═O)—-   (Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—    C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—    C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—    C(═O)-GGFG-NH—CH₂CH₂—O—CH₂— C(═O)—-   —CH₂— C(═O)—NH—CH₂CH₂— C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —CH₂— C(═O)—NH—CH₂CH₂— C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —CH₂— C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂— C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   —CH₂— C(═O)—NH—CH₂CH₂CH₂CH₂— C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —CH₂— C(═O)—NH—CH₂CH₂CH₂CH₂CH₂— C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   —C(═O)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—)-   —C(═O)—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—)-   —C(═O)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   —C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—    (N-ly-3-diminiccuS)-S—CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—    (N-ly-3-diminiccuS)-S—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—    (N-ly-3-diminiccuS)-S—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—    (N-ly-3-diminiccuS)-S—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—    (N-ly-3-diminiccuS)-S—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—    (N-ly-3-diminiccuS)-S—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—    (N-ly-3-diminiccuS)-S—CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—    (N-ly-3-diminiccuS)-S—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—    (N-ly-3-diminiccuS)-S—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—    (N-ly-3-diminiccuS)-S—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—    (N-ly-3-diminiccuS)-S—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)—S—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—.    [78] The linker according to [76], which is selected from the    following group, provided that the left terminal is a connecting    position with the antibody and the right terminal is a connecting    position with the antitumor compound:-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—.    [79] The linker according to [76], which is selected from the    following group, provided that the left terminal is a connecting    position with the antibody and the right terminal is a connecting    position with the antitumor compound:-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(—O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—.    [80] The linker according to [76], which is selected from the    following group, provided that the left terminal is a connecting    position with the antibody and the right terminal is a connecting    position with the antitumor compound:-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—.

Advantageous Effects of Invention

With an antibody-drug conjugate having an antitumor compound exatecanconjugated via a linker with a specific structure, an excellentantitumor effect and safety can be achieved.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an amino acid sequence of B7-H3 variant 1 (SEQ ID NO: 1).

FIG. 2 shows an amino acid sequence of B7-H3 variant 2 (SEQ ID NO: 2).

FIG. 3 shows an amino acid sequence of an M30-H1-type heavy chain (SEQID NO: 9).

FIG. 4 shows an amino acid sequence of an M30-H2-type heavy chain (SEQID NO: 10).

FIG. 5 shows an amino acid sequence of an M30-H3-type heavy chain (SEQID NO: 11).

FIG. 6 shows an amino acid sequence of an M30-H4-type heavy chain (SEQID NO: 12).

FIG. 7 shows an amino acid sequence of an M30-L1-type light chain (SEQID NO: 13).

FIG. 8 shows an amino acid sequence of an M30-L2-type light chain (SEQID NO: 14).

FIG. 9 shows an amino acid sequence of an M30-L3-type light chain (SEQID NO: 15).

FIG. 10 shows an amino acid sequence of an M30-L4-type light chain (SEQID NO: 16).

FIG. 11 shows an amino acid sequence of an M30-L5-type light chain (SEQID NO: 17).

FIG. 12 shows an amino acid sequence of an M30-L6-type light chain (SEQID NO: 18).

FIG. 13 shows an amino acid sequence of an M30-L7-type light chain (SEQID NO: 19).

FIG. 14 shows an amino acid sequence of an M30 antibody heavy chain (SEQID NO: 20).

FIG. 15 shows an amino acid sequence of an M30 antibody light chain (SEQID NO: 21).

FIG. 16 shows a nucleotide sequence of B7-H3 variant 1 (SEQ ID NO: 26).

FIG. 17 shows the effect of an antibody-drug conjugate (2) onsubcutaneously transplanted human melanoma line A375 cells. In thedrawing, the line with open rhombuses depicts results about untreatedtumor, the line with open triangles depicts the effect of an M30-H1-L4Pantibody, and the line with open circles depicts the effect of theantibody-drug conjugate (2).

FIG. 18 shows the effect of the antibody-drug conjugate (2) onsubcutaneously transplanted human melanoma line A375 cells. The linewith open rhombuses depicts results about untreated tumor, the line withfilled squares depicts the effect of the antibody-drug conjugate (2)administered at 0.1 mg/kg, the line with X marks depicts the effect ofthe antibody-drug conjugate (2) administered at 0.3 mg/kg, the line withfilled triangles depicts the effect of the antibody-drug conjugate (2)administered at 1 mg/kg, and the line with open circles depicts theeffect of the antibody-drug conjugate (2) administered at 3 mg/kg.

FIG. 19 shows the effect of the antibody-drug conjugate (2) onsubcutaneously transplanted human non-small cell lung cancer line Calu-6cells. The line with open rhombuses depicts results about untreatedtumor, the line with open triangles depicts the effect of an M30-H1-L4Pantibody, and the line with open circles depicts the effect of theantibody-drug conjugate (2).

FIG. 20 shows the effects of antibody-drug conjugates (1), (13), (41),and (55) on subcutaneously transplanted human melanoma line A375 cells.In the drawing, the line with open rhombuses depicts results aboutuntreated tumor, the line with open circles depicts the effect of theantibody-drug conjugate (1), the line with open triangles depicts theeffect of the antibody-drug conjugate (13), the line with X marksdepicts the effect of the antibody-drug conjugate (41), and the linewith open squares depicts the effect of the antibody-drug conjugate(55).

FIG. 21 shows the effects of antibody-drug conjugates (13), (41), and(55) on subcutaneously transplanted human non-small cell lung cancerline Calu-6 cells. The line with open rhombuses depicts results aboutuntreated tumor, the line with open circles depicts the effect ofDE-310, the line with open triangles depicts the effect of theantibody-drug conjugate (13), the line with X marks depicts the effectof the antibody-drug conjugate (41), and the line with open squaresdepicts the effect of the antibody-drug conjugate (55).

FIG. 22 shows the effects of antibody-drug conjugates (17), (18), (19),(59), (60), and (61) on subcutaneously transplanted human melanoma lineA375 cells. In the drawing, the line with filled rhombuses depictsresults about untreated tumor, the line with filled squares depicts theeffect of the antibody-drug conjugate (17), the line with open squaresdepicts the effect of the antibody-drug conjugate (18), the line withopen circles depicts the effect of the antibody-drug conjugate (19), theline with filled triangles depicts the effect of the antibody-drugconjugate (59), the line with open triangles depicts the effect of theantibody-drug conjugate (60), and the line with X marks depicts theeffect of the antibody-drug conjugate (61).

DESCRIPTION OF EMBODIMENTS

The antibody-drug conjugate of the present invention is an antitumordrug in which an antitumor antibody is conjugated to an antitumorcompound via a linker structure moiety and explained in detailhereinbelow.

[Antibody]

The antibody used in the antibody-drug conjugate of the presentinvention means an immunoglobulin and is a molecule containing anantigen-binding site immunospecifically binding to an antigen. The classof the antibody of the present invention may be any of IgG, IgE, IgM,IgD, IgA, and IgY and is preferably IgG. The subclass of the antibody ofthe present invention may be any of IgG1, IgG2, IgG3, IgG4, IgA1, andIgA2 and is preferably IgG1 or IgG2. The antibody may be derived fromany species, and preferred examples of the species can include humans,rats, mice, and rabbits. In case when derived from other than humanspecies, it is preferably chimerized or humanized using a well knowntechnique. The antibody of the present invention may be a polyclonalantibody or a monoclonal antibody and is preferably a monoclonalantibody.

The antibody of the present invention may be those which is capable oftargeting tumor cells. Since the antibody of the present invention isconjugated with a drug having antitumor activity via a linker, theantibody preferably possesses one or more of a property of recognizing atumor cell, a property of binding to a tumor cell, a property ofinternalizing in a tumor cell, and a property of damaging a tumor cell.

The binding activity of the antibody against tumor cells can beconfirmed using flow cytometry. The internalization of the antibody intotumor cells can be confirmed using (1) an assay of visualizing anantibody incorporated in cells under a fluorescence microscope using asecondary antibody (fluorescently labeled) binding to the therapeuticantibody (Cell Death and Differentiation (2008) 15, 751-761), (2) anassay of measuring the amount of fluorescence incorporated in cellsusing a secondary antibody (fluorescently labeled) binding to thetherapeutic antibody (Molecular Biology of the Cell, Vol. 15, 5268-5282,December 2004), or (3) a Mab-ZAP assay using an immunotoxin binding tothe therapeutic antibody wherein the toxin is released uponincorporation into cells to inhibit cell growth (Bio Techniques 28:162-165, January 2000).

The antitumor activity of the antibody refers to a cytotoxic activity orcytocidal effect against tumor cells and can be confirmed in vitro bydetermining inhibitory activity against cell growth. For example, acancer cell line overexpressing a target protein for the antibody iscultured, and the antibody is added at varying concentrations into theculture system to determin an inhibitory activity against focusformation, colony formation, and spheroid growth. The antitumor activitycan be confirmed in vivo, for example, by administering the antibody toa nude mouse with a transplanted tumor cell line highly expressing thetarget protein, and determining change in the cancer cell. Since thedrug conjugated in the antibody-drug conjugate exerts an antitumoreffect, it is more preferred but not essential that the antibody itselfshould have an antitumor effect. For exerting the antitumor effect andalso for specifically and selectively damaging tumor cells by the drug,it is important and also preferred that the antibody should have theproperty of internalizing to migrate into tumor cells.

Examples of such an antibody can include, but not limited to, ananti-A33 antibody, an anti-B7-H3 antibody, an anti-CanAg antibody, ananti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, ananti-CD33 antibody, an anti-CD56 antibody, an anti-CD70 antibody, ananti-CEA antibody, an anti-Cripto antibody, an anti-EphA2 antibody, ananti-G250 antibody, an anti-MUC1 antibody, an anti-GPNMB antibody, ananti-integrin antibody, an anti-PSMA antibody, an anti-tenascin-Cantibody, an anti-SLC44A4 antibody, and an anti-mesothelin antibody.

The antibody of the present invention is preferably an anti-CD30antibody, an anti-CD33 antibody, an anti-CD70 antibody, or an anti-B7-H3antibody, and more preferably an anti-B7-H3 antibody.

The antibody of the present invention can be obtained using a methodusually carried out in the art, which involves immunizing animals withan antigenic polypeptide and collecting and purifying antibodiesproduced in vivo. The origin of the antigen is not limited to humans,and the animals may be immunized with an antigen derived from anon-human animal such as a mouse, a rat and the like. In this case, thecross-reactivity of antibodies binding to the obtained heterologousantigen with human antigens can be tested to screen for an antibodyapplicable to a human disease.

Alternatively, antibody-producing cells which produce antibodies againstthe antigen are fused with myeloma cells according to a method known inthe art (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; andKennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N.Y.(1980)) to establish hybridomas, from which monoclonal antibodies can inturn be obtained.

The antigen can be obtained by genetically engineering host cells toproduce a gene encoding the antigenic protein. Specifically, vectorsthat permit expression of the antigen gene are prepared and transferredto host cells so that the gene is expressed. The antigen thus expressedcan be purified.

The anti-CD30 antibody, the anti-CD33 antibody, and the anti-CD70antibody can obtained by an approach known in the art with reference toWO2002/043661, U.S. Pat. No. 5,773,001, and WO2006/113909, respectively.

The B7-H3 antibody used in the present invention is preferably thosehaving properties as described below.

(1) An antibody having the following properties:

(a) specifically binding to B7-H3,

(b) having antibody-dependent cell-mediated phagocytosis (ADCP)activity, and

(c) having antitumor activity in vivo.

(2) The antibody according to (1), wherein B7-H3 is a moleculecomprising the amino acid sequence represented by SEQ ID NO: 1 or 2.(3) The antibody according to (1) or (2), wherein the antibody has CDRH1comprising the amino acid sequence represented by SEQ ID NO: 3, CDRH2comprising the amino acid sequence represented by SEQ ID NO: 4, andCDRH3 comprising the amino acid sequence represented by SEQ ID NO: 5 asheavy chain complementarity determining regions, and CDRL1 comprisingthe amino acid sequence represented by SEQ ID NO: 6, CDRL2 comprisingthe amino acid sequence represented by SEQ ID NO: 7, and CDRL3comprising the amino acid sequence represented by SEQ ID NO: 8 as lightchain complementarity determining regions.(4) The antibody according to any of (1) to (3), wherein the constantregion thereof is a human-derived constant region.(5) The antibody according to any of (1) to (4), wherein the antibody isa humanized antibody.(6) The antibody according to (5), wherein the antibody has a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of (a) an amino acid sequence described in aminoacid positions 20 to 141 in SEQ ID NO: 9, (b) an amino acid sequencedescribed in amino acid positions 20 to 141 in SEQ ID NO: 10, (c) anamino acid sequence described in amino acid positions 20 to 141 in SEQID NO: 11, (d) an amino acid sequence described in amino acid positions20 to 141 in SEQ ID NO: 12, (e) an amino acid sequence having at least95% or higher homology to any of the sequences (a) to (d), and (f) anamino acid sequence derived from any of the sequences (a) to (d) by thedeletions, replacements, or additions of at least one amino acid, and alight chain variable region comprising an amino acid sequence selectedfrom the group consisting of (g) an amino acid sequence described inamino acid positions 21 to 128 in SEQ ID NO: 13, (h) an amino acidsequence described in amino acid positions 21 to 128 in SEQ ID NO: 14,(i) an amino acid sequence described in amino acid positions 21 to 128in SEQ ID NO: 15, (j) an amino acid sequence described in amino acidpositions 21 to 128 in SEQ ID NO: 16, (k) an amino acid sequencedescribed in amino acid positions 21 to 128 in SEQ ID NO: 17, (1) anamino acid sequence described in amino acid positions 21 to 128 in SEQID NO: 18, (m) an amino acid sequence described in amino acid positions21 to 128 in SEQ ID NO: 19, (n) an amino acid sequence having at least95% or higher homology to any of the sequences (g) to (m), and (o) anamino acid sequence derived from any of the sequences (g) to (m) by thedeletions, replacements, or additions of at least one amino acid.(7) The antibody according to (6), wherein the antibody has a heavychain variable region and a light chain variable region selected fromthe group consisting of a heavy chain variable region comprising anamino acid sequence described in amino acid positions 20 to 141 in SEQID NO: 9 and a light chain variable region comprising an amino acidsequence described in amino acid positions 21 to 128 in SEQ ID NO: 13, aheavy chain variable region comprising an amino acid sequence describedin amino acid positions 20 to 141 in SEQ ID NO: 9 and a light chainvariable region comprising an amino acid sequence described in aminoacid positions to 128 in SEQ ID NO: 14, a heavy chain variable regioncomprising an amino acid sequence described in amino acid positions 20to 141 in SEQ ID NO: 9 and a light chain variable region comprising anamino acid sequence described in amino acid positions 21 to 128 in SEQID NO: 15, a heavy chain variable region comprising an amino acidsequence described in amino acid positions 20 to 141 in SEQ ID NO: 9 anda light chain variable region comprising an amino acid sequencedescribed in amino acid positions 21 to 128 in SEQ ID NO: 16, a heavychain variable region comprising an amino acid sequence described inamino acid positions to 141 in SEQ ID NO: 9 and a light chain variableregion comprising an amino acid sequence described in amino acidpositions 21 to 128 in SEQ ID NO: 17, a heavy chain variable regioncomprising an amino acid sequence described in amino acid positions 20to 141 in SEQ ID NO: 9 and a light chain variable region comprising anamino acid sequence described in amino acid positions 21 to 128 in SEQID NO: 18, a heavy chain variable region comprising an amino acidsequence described in amino acid positions 20 to 141 in SEQ ID NO: 9 anda light chain variable region comprising an amino acid sequencedescribed in amino acid positions to 128 in SEQ ID NO: 19, a heavy chainvariable region comprising an amino acid sequence described in aminoacid positions 20 to 141 in SEQ ID NO: 12 and a light chain variableregion comprising an amino acid sequence described in amino acidpositions 21 to 128 in SEQ ID NO: 13, a heavy chain variable regioncomprising an amino acid sequence described in amino acid positions 20to 141 in SEQ ID NO: 12 and a light chain variable region comprising anamino acid sequence described in amino acid positions 21 to 128 in SEQID NO: 14, a heavy chain variable region comprising an amino acidsequence described in amino acid positions 20 to 141 in SEQ ID NO: 12and a light chain variable region comprising an amino acid sequencedescribed in amino acid positions 21 to 128 in SEQ ID NO: 15, and aheavy chain variable region comprising an amino acid sequence describedin amino acid positions 20 to 141 in SEQ ID NO: 12 and a light chainvariable region comprising an amino acid sequence described in aminoacid positions 21 to 128 in SEQ ID NO: 16.(8) The antibody according to (6) or (7), wherein the antibody comprisesa heavy chain and a light chain selected from the group consisting of aheavy chain comprising an amino acid sequence described in amino acidpositions 20 to 471 in SEQ ID NO: 9 and a light chain comprising anamino acid sequence described in amino acid positions 21 to 233 in SEQID NO: 13, a heavy chain comprising an amino acid sequence described inamino acid positions 20 to 471 in SEQ ID NO: 9 and a light chaincomprising an amino acid sequence described in amino acid positions 21to 233 in SEQ ID NO: 14, a heavy chain comprising an amino acid sequencedescribed in amino acid positions 20 to 471 in SEQ ID NO: 9 and a lightchain comprising an amino acid sequence described in amino acidpositions 21 to 233 in SEQ ID NO: 15, a heavy chain comprising an aminoacid sequence described in amino acid positions 20 to 471 in SEQ ID NO:9 and a light chain comprising an amino acid sequence described in aminoacid positions 21 to 233 in SEQ ID NO: 16, a heavy chain comprising anamino acid sequence described in amino acid positions 20 to 471 in SEQID NO: 9 and a light chain comprising an amino acid sequence describedin amino acid positions 21 to 233 in SEQ ID NO: 17, a heavy chaincomprising an amino acid sequence described in amino acid positions 20to 471 in SEQ ID NO: 9 and a light chain comprising an amino acidsequence described in amino acid positions 21 to 233 in SEQ ID NO: 18, aheavy chain comprising an amino acid sequence described in amino acidpositions 20 to 471 in SEQ ID NO: 9 and a light chain comprising anamino acid sequence described in amino acid positions 21 to 233 in SEQID NO: 19, a heavy chain comprising an amino acid sequence described inamino acid positions 20 to 471 in SEQ ID NO: 12 and a light chaincomprising an amino acid sequence described in amino acid positions 21to 233 in SEQ ID NO: 13, a heavy chain comprising an amino acid sequencedescribed in amino acid positions 20 to 471 in SEQ ID NO: 12 and a lightchain comprising an amino acid sequence described in amino acidpositions 21 to 233 in SEQ ID NO: 14, a heavy chain comprising an aminoacid sequence described in amino acid positions 20 to 471 in SEQ ID NO:12 and a light chain comprising an amino acid sequence described inamino acid positions 21 to 233 in SEQ ID NO: 15, and a heavy chaincomprising an amino acid sequence described in amino acid positions 20to 471 in SEQ ID NO: 12 and a light chain comprising an amino acidsequence described in amino acid positions 21 to 233 in SEQ ID NO: 16.(9) The antibody according to any of (6) to (8), wherein the antibodycomprises a heavy chain and a light chain selected from the groupconsisting of a heavy chain comprising the amino acid sequencerepresented by SEQ ID NO: 9 and a light chain comprising the amino acidsequence represented by SEQ ID NO: 13, a heavy chain comprising theamino acid sequence represented by SEQ ID NO: 9 and a light chaincomprising the amino acid sequence represented by SEQ ID NO: 14, a heavychain comprising the amino acid sequence represented by SEQ ID NO: 9 anda light chain comprising the amino acid sequence represented by SEQ IDNO: 15, a heavy chain comprising the amino acid sequence represented bySEQ ID NO: 9 and a light chain comprising the amino acid sequencerepresented by SEQ ID NO: 16, a heavy chain comprising the amino acidsequence represented by SEQ ID NO: 9 and a light chain comprising theamino acid sequence represented by SEQ ID NO: 17, a heavy chaincomprising the amino acid sequence represented by SEQ ID NO: 9 and alight chain comprising the amino acid sequence represented by SEQ ID NO:18, a heavy chain comprising the amino acid sequence represented by SEQID NO: 9 and a light chain comprising the amino acid sequencerepresented by SEQ ID NO: 19, a heavy chain comprising the amino acidsequence represented by SEQ ID NO: 12 and a light chain comprising theamino acid sequence represented by SEQ ID NO: 13, a heavy chaincomprising the amino acid sequence represented by SEQ ID NO: 12 and alight chain comprising the amino acid sequence represented by SEQ ID NO:14, a heavy chain comprising the amino acid sequence represented by SEQID NO: 12 and a light chain comprising the amino acid sequencerepresented by SEQ ID NO: 15, and a heavy chain comprising the aminoacid sequence represented by SEQ ID NO: 12 and a light chain comprisingthe amino acid sequence represented by SEQ ID NO: 16.(10) The antibody according to (8) or (9), wherein the antibody lacks anamino acid at the carboxy terminus of the amino acid sequencerepresented by SEQ ID NO: 9 or 12 in the heavy chain.(11) An antibody obtained by a method for producing the antibodyaccording to any of (1) to (10), the method comprising the steps of:culturing a host cell transformed with an expression vector containing apolynucleotide encoding the antibody; and collecting the antibody ofinterest from the cultures obtained in the preceding step.(12) The antibody according to any of (1) to (11), wherein themodification of a glycan is regulated in order to enhanceantibody-dependent cytotoxic activity.

Hereinafter, the B7-H3 antibody used in the invention is described.

The terms “cancer” and “tumor” as used herein are used with the samemeaning.

The term “gene” as used herein includes not only DNA, but also mRNAthereof, cDNA thereof and cRNA thereof.

The term “polynucleotide” as used herein is used with the same meaningas a nucleic acid and also includes DNA, RNA, probes, oligonucleotides,and primers.

The terms “polypeptide” and “protein” as used herein are used withoutdistinction.

The term “cell” as used herein also includes cells in an animalindividual and cultured cells.

The term “B7-H3” as used herein is used in the same meaning as B7-H3protein, and also refers to B7-H3 variant 1 and/or B7-H3 variant 2.

The term “CDR” as used herein refers to a complementarity determiningregion (CDR), and it is known that each heavy and light chain of anantibody molecule has three complementarity determining regions (CDRs).The CDR is also called the hypervariable region, and is present in avariable region of each heavy and light chain of an antibody. It is asite which has unusually high variability in its primary structure, andthere are three separate CDRs in the primary structure of each heavy andlight polypeptide chain. In this specification, as for the CDRs of anantibody, the CDRs of the heavy chain are represented by CDRH1, CDRH2,and CDRH3 from the amino-terminal side of the amino acid sequence of theheavy chain, and the CDRs of the light chain are represented by CDRL1,CDRL2, and CDRL3 from the amino-terminal side of the amino acid sequenceof the light chain. These sites are proximate to one another in thetertiary structure and determine the specificity for an antigen to whichthe antibody binds.

The phrase “hybridization is performed under stringent conditions” asused herein refers to a process in which hybridization is performedunder conditions under which identification can be achieved byperforming hybridization at 68° C. in a commercially availablehybridization solution ExpressHyb Hybridization Solution (manufacturedby Clontech, Inc.) or by performing hybridization at 68° C. in thepresence of 0.7 to 1.0 M NaCl using a filter having DNA immobilizedthereon, followed by performing washing at 68° C. using 0.1 to 2×SSCsolution (1×SSC solution is composed of 150 mM NaCl and 15 mM sodiumcitrate) or under conditions equivalent thereto.

1. B7-H3

B7-H3 is a member of the B7 family expressed on antigen-presenting cellsas a co-stimulatory molecule, and is considered to act on a receptor onT cells to enhance or suppress immune activity.

B7-H3 is a protein having a single-pass transmembrane structure, and theN-terminal extracellular domain of B7-H3 contains two variants. TheB7-H3 variant 1 (4Ig-B7-H3) contains a V-like or C-like Ig domain at twosites, respectively, and the B7-H3 variant 2 (2Ig-B7-H3) contains aV-like or C-like Ig domain at one site, respectively.

As for B7-H3 to be used in the invention, B7-H3 can be directly purifiedfrom B7-H3-expressing cells of a human or a non-human mammal (such as arat or a mouse) and used, or a cell membrane fraction of theabove-described cells can be prepared and used. Further, B7-H3 can beobtained by in vitro synthesis thereof or production thereof in a hostcell through genetic engineering. In the genetic engineering,specifically, after B7-H3 cDNA is integrated into a vector capable ofexpressing B7-H3 cDNA, B7-H3 can be obtained by synthesizing it in asolution containing an enzyme, a substrate and an energy substancerequired for transcription and translation, or by expressing B7-H3 inanother prokaryotic or eucaryotic transformed host cell.

The amino acid sequence of an open reading frame (ORF) of a human B7-H3variant 1 gene is represented by SEQ ID NO: 1 in the Sequence Listing.Further, the sequence of SEQ ID NO: 1 is shown in FIG. 1.

The amino acid sequence of an ORF of a human B7-H3 variant 2 gene isrepresented by SEQ ID NO: 2 in the Sequence Listing. Further, thesequence of SEQ ID NO: 2 is shown in FIG. 2.

Further, a protein which consists of an amino acid sequence wherein oneor several amino acids are substituted, deleted and/or added in any ofthe above-described amino acid sequences of B7-H3 and also has abiological activity equivalent to that of the protein is also includedin B7-H3.

Mature human B7-H3 variant 1 from which the signal sequence has beenremoved corresponds to an amino acid sequence consisting of amino acidresidues 27 to 534 of the amino acid sequence represented by SEQ IDNO: 1. Further, mature human B7-H3 variant 2 from which the signalsequence has been removed corresponds to an amino acid sequenceconsisting of amino acid residues 27 to 316 of the amino acid sequencerepresented by SEQ ID NO: 2.

2. Production of Anti-B7-H3 Antibody

The antibody against B7-H3 of the invention can be obtained byimmunizing an animal with B7-H3 or an arbitrary polypeptide selectedfrom the amino acid sequence of B7-H3, and collecting and purifying theantibody produced in vivo according to a common procedure. Thebiological species of B7-H3 to be used as an antigen is not limited tobeing human, and an animal can be immunized with B7-H3 derived from ananimal other than humans such as a mouse or a rat. In this case, byexamining the cross-reactivity between an antibody binding to theobtained heterologous B7-H3 and human B7-H3, an antibody applicable to ahuman disease can be selected.

Further, a monoclonal antibody can be obtained from a hybridomaestablished by fusing antibody-producing cells which produce an antibodyagainst B7-H3 with myeloma cells according to a known method (forexample, Kohler and Milstein, Nature, (1975) 256, pp. 495-497; Kennet,R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, N.Y. (1980)).

B7-H3 to be used as an antigen can be obtained by expressing B7-H3 genein a host cell using genetic enginering.

Specifically, a vector capable of expressing B7-H3 gene is produced, andthe resulting vector is transfected into a host cell to express thegene, and then, the expressed B7-H3 is purified. Hereinafter, a methodof obtaining an antibody against B7-H3 is specifically described.

(1) Preparation of Antigen

Examples of the antigen to be used for producing the anti-B7-H3 antibodyinclude B7-H3, a polypeptide consisting of a partial amino acid sequencecomprising at least 6 consecutive amino acids of B7-H3, and a derivativeobtained by adding a given amino acid sequence or carrier thereto.

B7-H3 can be purified directly from human tumor tissues or tumor cellsand used. Further, B7-H3 can be obtained by synthesizing it in vitro orby producing it in a host cell by genetic engineering.

With respect to the genetic engineering, specifically, after B7-H3 cDNAis integrated into a vector capable of expressing B7-H3 cDNA, B7-H3 canbe obtained by synthesizing it in a solution containing an enzyme, asubstrate and an energy substance required for transcription andtranslation, or by expressing B7-H3 in another prokaryotic or eucaryotictransformed host cell.

Further, the antigen can also be obtained as a secretory protein byexpressing a fusion protein obtained by ligating the extracellulardomain of B7-H3, which is a membrane protein, to the constant region ofan antibody in an appropriate host-vector system.

B7-H3 cDNA can be obtained by, for example, a so-called PCR method inwhich a polymerase chain reaction (hereinafter referred to as “PCR”) isperformed using a cDNA library expressing B7-H3 cDNA as a template andprimers which specifically amplify B7-H3 cDNA (see Saiki, R. K., et al.,Science, (1988) 239, pp. 487-489).

As the in vitro synthesis of the polypeptide, for example, RapidTranslation System (RTS) manufactured by Roche Diagnostics, Inc. can beexemplified, but it is not limited thereto.

Examples of the prokaryotic host cells include Escherichia coli andBacillus subtilis. In order to transform the host cells with a targetgene, the host cells are transformed by a plasmid vector comprising areplicon, i.e., a replication origin derived from a species compatiblewith the host, and a regulatory sequence. Further, the vector preferablyhas a sequence capable of imposing phenotypic selectivity on thetransformed cell.

Examples of the eucaryotic host cells include vertebrate cells, insectcells, and yeast cells. As the vertebrate cells, for example, simian COScells (Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCC CRL-1650), murinefibroblasts NIH3T3 (ATCC No. CRL-1658), and dihydrofolatereductase-deficient strains (Urlaub, G. and Chasin, L. A., Proc. Natl.Acad. Sci. USA (1980) 77, pp. 4126-4220) of Chinese hamster ovariancells (CHO cells; ATCC: CCL-61); and the like are often used, however,the cells are not limited thereto.

The thus obtained transformant can be cultured according to a commonprocedure, and by the culturing of the transformant, a targetpolypeptide is produced intracellularly or extracellularly.

A suitable medium to be used for the culturing can be selected fromvarious commonly used culture media depending on the employed hostcells. If Escherichia coli is employed, for example, an LB mediumsupplemented with an antibiotic such as ampicillin or IPMG as needed canbe used.

A recombinant protein produced intracellularly or extracellularly by thetransformant through such culturing can be separated and purified by anyof various known separation methods utilizing the physical or chemicalproperty of the protein.

Specific examples of the methods include treatment with a common proteinprecipitant, ultrafiltration, various types of liquid chromatographysuch as molecular sieve chromatography (gel filtration), adsorptionchromatography, ion exchange chromatography, and affinitychromatography, dialysis, and a combination thereof.

Further, by attaching a tag of six histidine residues to a recombinantprotein to be expressed, the protein can be efficiently purified with anickel affinity column. Alternatively, by attaching the IgG Fc region toa recombinant protein to be expressed, the protein can be efficientlypurified with a protein A column.

By combining the above-described methods, a large amount of a targetpolypeptide can be easily produced in high yield and high purity.

(2) Production of Anti-B7-H3 Monoclonal Antibody

Examples of the antibody specific binding to B7-H3 include a monoclonalantibody specific binding to B7-H3, and a method of obtaining theantibody is as described below.

The production of a monoclonal antibody generally requires the followingoperational steps of:

(a) purifying a biopolymer to be used as an antigen;

(b) preparing antibody-producing cells by immunizing an animal byinjection of the antigen, collecting the blood, assaying its antibodytiter to determine when the spleen is excised;

(c) preparing myeloma cells (hereinafter referred to as “myeloma”);

(d) fusing the antibody-producing cells with the myeloma;

(e) screening a group of hybridomas producing a desired antibody;

(f) dividing the hybridomas into single cell clones (cloning);

(g) optionally, culturing the hybridoma or rearing an animal implantedwith the hybridoma for producing a large amount of a monoclonalantibody;

(h) examining the thus produced monoclonal antibody for biologicalactivity and binding specificity, or assaying the same for properties asa labeled reagent; and the like.

Hereinafter, the method of producing a monoclonal antibody will bedescribed in detail following the above steps, however, the method isnot limited thereto, and, for example, antibody-producing cells otherthan spleen cells and myeloma can be used.

(a) Purification of Antigen

As the antigen, B7-H3 prepared by the method as described above or apartial peptide thereof can be used.

Further, a membrane fraction prepared from recombinant cells expressingB7-H3 or the recombinant cells expressing B7-H3 themselves, and also apartial peptide of the protein of the invention chemically synthesizedby a method known to those skilled in the art can also be used as theantigen.

(b) Preparation of Antibody-Producing Cells

The antigen obtained in the step (a) is mixed with an adjuvant such asFreund's complete or incomplete adjuvant or aluminum potassium sulfateand the resulting mixture is used as an immunogen to immunize anexperimental animal. As the experimental animal, any animal used in aknown hybridoma production method can be used without any trouble.Specifically, for example, a mouse, a rat, a goat, sheep, cattle, ahorse, or the like can be used. However, from the viewpoint of ease ofavailability of myeloma cells to be fused with the extractedantibody-producing cells, a mouse or a rat is preferably used as theanimal to be immunized.

Further, the strain of a mouse or a rat to be used is not particularlylimited, and in the case of a mouse, for example, various strains suchas A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA, FL, HTH,HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, and 129 and the like can beused, and in the case of a rat, for example, Wistar, Low, Lewis,Sprague, Dawley, ACI, BN, Fischer and the like can be used.

These mice and rats are commercially available frombreeders/distributors of experimental animals, for example, CLEA Japan,Inc. and Charles River Laboratories Japan, Inc.

Among these, in consideration of compatibility of fusing with myelomacells described below, in the case of a mouse, BALB/c strain, and in thecase of a rat, Wistar and Low strains are particularly preferred as theanimal to be immunized.

Further, in consideration of antigenic homology between humans and mice,it is also preferred to use a mouse having decreased biological functionto remove auto-antibodies, that is, a mouse with an autoimmune disease.

The age of such mouse or rat at the time of immunization is preferably 5to 12 weeks of age, more preferably 6 to 8 weeks of age.

In order to immunize an animal with B7-H3 or a recombinant thereof, forexample, a known method described in detail in, for example, Weir, D.M., Handbook of Experimental Immunology Vol. I. II. III., BlackwellScientific Publications, Oxford (1987), Kabat, E. A. and Mayer, M. M.,Experimental Immunochemistry, Charles C Thomas Publisher Springfield,Ill. (1964) or the like can be used.

Among these immunization methods, a preferred specific method in theinvention is, for example, as follows.

That is, first, a membrane protein fraction serving as the antigen orcells caused to express the antigen is/are intradermally orintraperitoneally administrated to an animal.

However, the combination of both routes of administration is preferredfor increasing the immunization efficiency, and when intradermaladministration is performed in the first half and intraperitonealadministration is performed in the latter half or only at the lastdosing, the immunization efficiency can be particularly increased.

The administration schedule of the antigen varies depending on the typeof animal to be immunized, individual difference or the like. However,in general, an administration schedule in which the frequency ofadministration of the antigen is 3 to 6 times and the dosing interval is2 to 6 weeks is preferred, and an administration schedule in which thefrequency of administration of the antigen is 3 to 4 times and thedosing interval is 2 to 4 weeks is more preferred.

Further, the dose of the antigen varies depending on the type of animal,individual differences or the like, however, the dose is generally setto 0.05 to 5 mg, preferably about 0.1 to 0.5 mg.

A booster immunization is performed 1 to 6 weeks, preferably 2 to 4weeks, more preferably 2 to 3 weeks after the administration of theantigen as described above.

The dose of the antigen at the time of performing the boosterimmunization varies depending on the type or size of animal or the like,however, in the case of, for example, a mouse, the dose is generally setto 0.05 to 5 mg, preferably 0.1 to 0.5 mg, more preferably about 0.1 to0.2 mg.

Spleen cells or lymphocytes including antibody-producing cells areaseptically removed from the immunized animal 1 to 10 days, preferably 2to 5 days, more preferably 2 to 3 days after the booster immunization.At this time, the antibody titer is measured, and if an animal having asufficiently increased antibody titer is used as a supply source of theantibody-producing cells, the subsequent procedure can be carried outmore efficiently.

Examples of the method of measuring the antibody titer to be used hereinclude an RIA method and an ELISA method, but the method is not limitedthereto.

For example, if an ELISA method is employed, the measurement of theantibody titer in the invention can be carried out according to theprocedures as described below.

First, a purified or partially purified antigen is adsorbed to thesurface of a solid phase such as a 96-well plate for ELISA, and thesurface of the solid phase having no antigen adsorbed thereto is coveredwith a protein unrelated to the antigen such as bovine serum albumin(hereinafter referred to as “BSA”). After washing the surface, thesurface is brought into contact with a serially-diluted sample (forexample, mouse serum) as a primary antibody to allow the antibody in thesample to bind to the antigen.

Further, as a secondary antibody, an antibody labeled with an enzymeagainst a mouse antibody is added and is allowed to bind to the mouseantibody. After washing, a substrate for the enzyme is added and achange in absorbance which occurs due to color development induced bydegradation of the substrate or the like is measured and the antibodytiter is calculated based on the measurement.

The separation of the antibody-producing cells from the spleen cells orlymphocytes of the immunized animal can be carried out according to aknown method (for example, Kohler et al., Nature (1975), 256, p. 495;Kohler et al., Eur. J. Immunol. (1977), 6, p. 511; Milstein et al.,Nature (1977), 266, p. 550; Walsh, Nature (1977), 266, p. 495). Forexample, in the case of spleen cells, a general method in which theantibody-producing cells are separated by homogenizing the spleen toobtain the cells through filtration with a stainless steel mesh andsuspending the cells in Eagle's Minimum Essential Medium (MEM) can beemployed.

(c) Preparation of Myeloma Cells (Hereinafter Referred to as “Myeloma”)

The myeloma cells to be used for cell fusion are not particularlylimited and suitable cells can be selected from known cell lines.However, in consideration of convenience when a hybridoma is selectedfrom fused cells, it is preferred to use an HGPRT (hypoxanthine-guaninephosphoribosyl transferase) deficient strain whose selection procedurehas been established.

More specifically, examples of the HGPRT-deficient strain includeX63-Ag8(X63), NS1-ANS/1(NS1), P3X63-Ag8.U1(P3U1), X63-Ag8.653(X63.653),SP2/0-Ag14(SP2/0), MPC11-45.6TG1.7(45.6TG), FO, S149/5XXO, and BU.1derived from mice; 210.RSY3.Ag.1.2.3(Y3) derived from rats; andU266AR(SKO-007), GM1500-GTG-Al2(GM1500), UC729-6, LICR-LOW-HMy2(HMy2)and 8226AR/NIP4-1(NP41) derived from humans. These HGPRT-deficientstrains are available from, for example, the American Type CultureCollection (ATCC) or the like.

These cell strains are subcultured in an appropriate medium such as an8-azaguanine medium [a medium obtained by adding 8-azaguanine to an RPMI1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin,and fetal calf serum (hereinafter referred to as “FCS”)], Iscove'sModified Dulbecco's Medium (hereinafter referred to as “IMDM”), orDulbecco's Modified Eagle Medium (hereinafter referred to as “DMEM”). Inthis case, 3 to 4 days before performing cell fusion, the cells aresubcultured in a normal medium [for example, an ASF104 medium(manufactured by Ajinomoto Co., Ltd.) containing 10% FCS] to ensure notless than 2×10⁷ cells on the day of cell fusion.

(d) Cell Fusion

Fusion between the antibody-producing cells and the myeloma cells can beappropriately performed according to a known method (Weir, D. M.Handbook of Experimental Immunology Vol. I. II. III., BlackwellScientific Publications, Oxford (1987), Kabat, E. A. and Mayer, M. M.,Experimental Immunochemistry, Charles C Thomas Publisher, Springfield,Ill. (1964), etc.), under conditions such that the survival rate ofcells is not excessively reduced.

As such a method, for example, a chemical method in which theantibody-producing cells and the myeloma cells are mixed in a solutioncontaining a polymer such as polyethylene glycol at a highconcentration, a physical method using electric stimulation, or the likecan be used. Among these methods, a specific example of the chemicalmethod is as described below.

That is, in the case where polyethylene glycol is used in the solutioncontaining a polymer at a high concentration, the antibody-producingcells and the myeloma cells are mixed in a solution of polyethyleneglycol having a molecular weight of 1500 to 6000, more preferably 2000to 4000 at a temperature of from 30 to 40° C., preferably from 35 to 38°C. for 1 to 10 minutes, preferably 5 to 8 minutes.

(e) Selection of a Group of Hybridomas

The method of selecting hybridomas obtained by the above-described cellfusion is not particularly limited. Usually, an HAT (hypoxanthine,aminopterin, thymidine) selection method (Kohler et al., Nature (1975),256, p. 495; Milstein et al., Nature (1977), 266, p. 550) is used.

This method is effective when hybridomas are obtained using the myelomacells of an HGPRT-deficient strain which cannot survive in the presenceof aminopterin.

That is, by culturing unfused cells and hybridomas in an HAT medium,only hybridomas resistant to aminopterin are selectively allowed tosurvive and proliferate.

(f) Division into Single Cell Clone (Cloning)

As a cloning method for hybridomas, a known method such as amethylcellulose method, a soft agarose method, or a limiting dilutionmethod can be used (see, for example, Barbara, B. M. and Stanley, M. S.:Selected Methods in Cellular Immunology, W. H. Freeman and Company, SanFrancisco (1980)). Among these methods, particularly, athree-dimensional culture method such as a methylcellulose method ispreferred. For example, the group of hybridomas produced by cell fusionare suspended in a methylcellulose medium such as ClonaCell-HY SelectionMedium D (manufactured by StemCell Technologies, inc., #03804) andcultured. Then, the formed hybridoma colonies are collected, wherebymonoclonal hybridomas can be obtained. The collected respectivehybridoma colonies are cultured, and a hybridoma which has beenconfirmed to have a stable antibody titer in an obtained hybridomaculture supernatant is selected as a B7-H3 monoclonal antibody-producinghybridoma strain.

Examples of the thus established hybridoma strain include B7-H3hybridoma M30. In this specification, an antibody produced by the B7-H3hybridoma M30 is referred to as “M30 antibody” or simply “M30”.

The heavy chain of the M30 antibody has an amino acid sequencerepresented by SEQ ID NO: 20 in the Sequence Listing. Further, the lightchain of the M30 antibody has an amino acid sequence represented by SEQID NO: 21 in the Sequence Listing. In the heavy chain amino acidsequence represented by SEQ ID NO: 20 in the Sequence Listing, an aminoacid sequence consisting of amino acid residues 1 to 19 is a signalsequence, an amino acid sequence consisting of amino acid residues to141 is a variable region, and an amino acid sequence consisting of aminoacid residues 142 to 471 is a constant region. Further, in the lightchain amino acid sequence represented by SEQ ID NO: 21 in the SequenceListing, an amino acid sequence consisting of amino acid residues 1 to22 is a signal sequence, an amino acid sequence consisting of amino acidresidues to 130 is a variable region, and an amino acid sequenceconsisting of amino acid residues 131 to 235 is a constant region.

(g) Preparation of Monoclonal Antibody by Culturing Hybridoma

By culturing the thus selected hybridoma, a monoclonal antibody can beefficiently obtained. However, prior to culturing, it is preferred toperform screening of a hybridoma which produces a target monoclonalantibody.

In such screening, a known method can be employed.

The measurement of the antibody titer in the invention can be carriedout by, for example, an ELISA method explained in item (b) describedabove.

The hybridoma obtained by the method described above can be stored in afrozen state in liquid nitrogen or in a freezer at −80° C. or below.

After completion of cloning, the medium is changed from an HT medium toa normal medium, and the hybridoma is cultured.

Large-scale culture is performed by rotation culture using a largeculture bottle or by spinner culture. From the supernatant obtained bythe large-scale culture, a monoclonal antibody which specifically bindsto the protein of the invention can be obtained by purification using amethod known to those skilled in the art such as gel filtration.

Further, the hybridoma is injected into the abdominal cavity of a mouseof the same strain as the hybridoma (for example, the above-describedBALB/c) or a Nu/Nu mouse to proliferate the hybridoma, whereby theascites containing a large amount of the monoclonal antibody of theinvention can be obtained.

In the case where the hybridoma is administrated in the abdominalcavity, if a mineral oil such as 2,6,10,14-tetramethyl pentadecane(pristane) is administrated 3 to 7 days prior thereto, a larger amountof the ascites can be obtained.

For example, an immunosuppressant is previously injected into theabdominal cavity of a mouse of the same strain as the hybridoma toinactivate T cells. 20 days thereafter, 10⁶ to 10⁷ hybridoma clone cellsare suspended in a serum-free medium (0.5 ml), and the suspension isadministrated in the abdominal cavity of the mouse. In general, when theabdomen is expanded and filled with the ascites, the ascites iscollected from the mouse. By this method, the monoclonal antibody can beobtained at a concentration which is about 100 times or much higher thanthat in the culture solution.

The monoclonal antibody obtained by the above-described method can bepurified by a method described in, for example, Weir, D. M.: Handbook ofExperimental Immunology Vol. I, II, III, Blackwell ScientificPublications, Oxford (1978).

The thus obtained monoclonal antibody has high antigen specificity forB7-H3.

(h) Assay of Monoclonal Antibody

The isotype and subclass of the thus obtained monoclonal antibody can bedetermined as follows.

First, examples of the identification method include an Ouchterlonymethod, an ELISA method, and an RIA method.

An Ouchterlony method is simple, but when the concentration of themonoclonal antibody is low, a condensation operation is required.

On the other hand, when an ELISA method or an RIA method is used, bydirectly reacting the culture supernatant with an antigen-adsorbed solidphase and using antibodies corresponding to various types ofimmunoglobulin isotypes and subclasses as secondary antibodies, theisotype and subclass of the monoclonal antibody can be identified.

In addition, as a simpler method, a commercially availableidentification kit (for example, Mouse Typer Kit manufactured by Bio-RadLaboratories, Inc.) or the like can also be used.

Further, the quantitative determination of a protein can be performed bythe Folin Lowry method and a method of calculation based on theabsorbance at 280 nm [1.4 (OD 280)=Immunoglobulin 1 mg/ml].

Further, even when the monoclonal antibody is separately andindependently obtained by performing again the steps of (a) to (h) in(2), it is possible to obtain an antibody having a cytotoxic activityequivalent to that of the M30 antibody. As one example of such anantibody, an antibody which binds to the same epitope as the M30antibody can be exemplified. The M30 recognizes an epitope in the IgC1or IgC2 domain, which is a domain in the B7-H3 extracellular domain, andbinds to the IgC1 domain or the IgC2 domain or both. Therefore, as theepitope for the antibody of the invention, particularly, an epitopepresent in the IgC1 or IgC2 domain of B7-H3 can be exemplified. If anewly produced monoclonal antibody binds to a partial peptide or apartial tertiary structure to which the M30 antibody binds, it can bedetermined that the monoclonal antibody binds to the same epitope as theM30 antibody. Further, by confirming that the monoclonal antibodycompetes with the M30 antibody for the binding to B7-H3 (that is, themonoclonal antibody inhibits the binding between the M30 antibody andB7-H3), it can be determined that the monoclonal antibody binds to thesame epitope as the M30 antibody even if the specific epitope sequenceor structure has not been determined. When it is confirmed that themonoclonal antibody binds to the same epitope as the M30 antibody, themonoclonal antibody is strongly expected to have a cytotoxic activityequivalent to that of the M30 antibody.

(3) Other Antibodies

The antibody of the invention includes not only the above-describedmonoclonal antibody against B7-H3 but also a recombinant antibodyobtained by artificial modification for the purpose of decreasingheterologous antigenicity to humans such as a chimeric antibody, ahumanized antibody and a human antibody. These antibodies can beproduced using a known method.

As the chimeric antibody, an antibody in which antibody variable andconstant regions are derived from different species, for example, achimeric antibody in which a mouse- or rat-derived antibody variableregion is connected to a human-derived antibody constant region can beexemplified (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

As the humanized antibody, an antibody obtained by integrating only acomplementarity determining region (CDR) into a human-derived antibody(see Nature (1986) 321, pp. 522-525), and an antibody obtained bygrafting a part of the amino acid residues of the framework as well asthe CDR sequence to a human antibody by a CDR-grafting method (WO90/07861) can be exemplified.

However, the humanized antibody derived from the M30 antibody is notlimited to a specific humanized antibody as long as the humanizedantibody has all 6 types of CDR sequences of the M30 antibody and has anantitumor activity. The heavy chain variable region of the M30 antibodyhas CDRH1 (NYVMH) consisting of an amino acid sequence represented bySEQ ID NO: 3 in the Sequence Listing, CDRH2 (YINPYNDDVKYNEKFKG)consisting of an amino acid sequence represented by SEQ ID NO: 4 in theSequence Listing, and CDRH3 (WGYYGSPLYYFDY) consisting of an amino acidsequence represented by SEQ ID NO: 5 in the Sequence Listing. Further,the light chain variable region of the M30 antibody has CDRL1(RASSRLIYMH) consisting of an amino acid sequence represented by SEQ IDNO: 6 in the Sequence Listing, CDRL2 (ATSNLAS) consisting of an aminoacid sequence represented by SEQ ID NO: 7 in the Sequence Listing, andCDRL3 (QQWNSNPPT) consisting of an amino acid sequence represented bySEQ ID NO: 8 in the Sequence Listing.

As an example of the humanized antibody of a mouse antibody M30, anarbitrary combination of a heavy chain comprising a heavy chain variableregion consisting of any one of (1) an amino acid sequence consisting ofamino acid residues 20 to 141 of SEQ ID NO: 9, 10, 11, or 12 in theSequence Listing, (2) an amino acid sequence having a homology of atleast 95% or more with the amino acid sequence (1) described above, and(3) an amino acid sequence wherein one or several amino acids in theamino acid sequence (1) described above are deleted, substituted oradded and a light chain comprising a light chain variable regionconsisting of any one of (4) an amino acid sequence consisting of aminoacid residues 21 to 128 of SEQ ID NO: 13, 14, 15, 16, 17, 18, or 19 inthe Sequence Listing, (5) an amino acid sequence having a homology of atleast 95% or more with the amino acid sequence (4) described above, and(6) an amino acid sequence wherein one or several amino acids in theamino acid sequence (4) described above are deleted, substituted oradded can be exemplified.

The term “several” as used herein refers to 1 to 10, 1 to 9, 1 to 8, 1to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

As the amino acid substitution in this specification, a conservativeamino acid substitution is preferred. The conservative amino acidsubstitution refers to a substitution occurring within a group of aminoacids related to amino acid side chains. Preferred amino acid groups areas follows: an acidic group (aspartic acid and glutamic acid); a basicgroup (lysine, arginine, and histidine); a non-polar group (alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine, andtryptophan); and an uncharged polar family (glycine, asparagine,glutamine, cysteine, serine, threonine, and tyrosine). More preferredamino acid groups are as follows: an aliphatic hydroxy group (serine andthreonine); an amide-containing group (asparagine and glutamine); analiphatic group (alanine, valine, leucine, and isoleucine); and anaromatic group (phenylalanine, tryptophan, and tyrosine). Such an aminoacid substitution is preferably performed within a range which does notimpair the properties of a substance having the original amino acidsequence.

As an antibody which has a preferred combination of a heavy chain and alight chain described above, an antibody consisting of a heavy chaincomprising a heavy chain variable region consisting of an amino acidsequence consisting of amino acid residues 20 to 141 of SEQ ID NO: 9 anda light chain comprising a light chain variable region consisting of anamino acid sequence consisting of amino acid residues 21 to 128 of SEQID NO: 13; an antibody consisting of a heavy chain comprising a heavychain variable region consisting of an amino acid sequence consisting ofamino acid residues 20 to 141 of SEQ ID NO: 9 and a light chaincomprising a light chain variable region consisting of an amino acidsequence consisting of amino acid residues 21 to 128 of SEQ ID NO: 14;an antibody consisting of a heavy chain comprising a heavy chainvariable region consisting of an amino acid sequence consisting of aminoacid residues 20 to 141 of SEQ ID NO: 9 and a light chain comprising alight chain variable region consisting of an amino acid sequenceconsisting of amino acid residues 21 to 128 of SEQ ID NO: 15; anantibody consisting of a heavy chain comprising a heavy chain variableregion consisting of an amino acid sequence consisting of amino acidresidues 20 to 141 of SEQ ID NO: 9 and a light chain comprising a lightchain variable region consisting of an amino acid sequence consisting ofamino acid residues 21 to 128 of SEQ ID NO: 16; an antibody consistingof a heavy chain comprising a heavy chain variable region consisting ofan amino acid sequence consisting of amino acid residues 20 to 141 ofSEQ ID NO: 9 and a light chain comprising a light chain variable regionconsisting of an amino acid sequence consisting of amino acid residues21 to 128 of SEQ ID NO: 17; an antibody consisting of a heavy chaincomprising a heavy chain variable region consisting of an amino acidsequence consisting of amino acid residues 20 to 141 of SEQ ID NO: 9 anda light chain comprising a light chain variable region consisting of anamino acid sequence consisting of amino acid residues 21 to 128 of SEQID NO: 18; an antibody consisting of a heavy chain comprising a heavychain variable region consisting of an amino acid sequence consisting ofamino acid residues 20 to 141 of SEQ ID NO: 9 and a light chaincomprising a light chain variable region consisting of an amino acidsequence consisting of amino acid residues 21 to 128 of SEQ ID NO: 19;an antibody consisting of a heavy chain comprising a heavy chainvariable region consisting of an amino acid sequence consisting of aminoacid residues 20 to 141 of SEQ ID NO: 12 and a light chain comprising alight chain variable region consisting of an amino acid sequenceconsisting of amino acid residues 21 to 128 of SEQ ID NO: 13; anantibody consisting of a heavy chain comprising a heavy chain variableregion consisting of an amino acid sequence consisting of amino acidresidues 20 to 141 of SEQ ID NO: 12 and a light chain comprising a lightchain variable region consisting of an amino acid sequence consisting ofamino acid residues 21 to 128 of SEQ ID NO: 14; an antibody consistingof a heavy chain comprising a heavy chain variable region consisting ofan amino acid sequence consisting of amino acid residues 20 to 141 ofSEQ ID NO: 12 and a light chain comprising a light chain variable regionconsisting of an amino acid sequence consisting of amino acid residues21 to 128 of SEQ ID NO: 15; and an antibody consisting of a heavy chaincomprising a heavy chain variable region consisting of an amino acidsequence consisting of amino acid residues 20 to 141 of SEQ ID NO: 12and a light chain comprising a light chain variable region consisting ofan amino acid sequence consisting of amino acid residues 21 to 128 ofSEQ ID NO: 16 can be exemplified.

Further, as an antibody which has a more preferred combination of aheavy chain and a light chain described above, an antibody consisting ofa heavy chain consisting of an amino acid sequence consisting of aminoacid residues 20 to 471 of SEQ ID NO: 9 and a light chain consisting ofan amino acid sequence consisting of amino acid residues 21 to 233 ofSEQ ID NO: 13; an antibody consisting of a heavy chain consisting of anamino acid sequence consisting of amino acid residues 20 to 471 of SEQID NO: 9 and a light chain consisting of an amino acid sequenceconsisting of amino acid residues 21 to 233 of SEQ ID NO: 14; anantibody consisting of a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 9 anda light chain consisting of an amino acid sequence consisting of aminoacid residues 21 to 233 of SEQ ID NO: 15; an antibody consisting of aheavy chain consisting of an amino acid sequence consisting of aminoacid residues 20 to 471 of SEQ ID NO: 9 and a light chain consisting ofan amino acid sequence consisting of amino acid residues 21 to 233 ofSEQ ID NO: 16; an antibody consisting of a heavy chain consisting of anamino acid sequence consisting of amino acid residues 20 to 471 of SEQID NO: 9 and a light chain consisting of an amino acid sequenceconsisting of amino acid residues 21 to 233 of SEQ ID NO: 17; anantibody consisting of a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 9 anda light chain consisting of an amino acid sequence consisting of aminoacid residues 21 to 233 of SEQ ID NO: 18; an antibody consisting of aheavy chain consisting of an amino acid sequence consisting of aminoacid residues 20 to 471 of SEQ ID NO: 9 and a light chain consisting ofan amino acid sequence consisting of amino acid residues 21 to 233 ofSEQ ID NO: 19; an antibody consisting of a heavy chain consisting of anamino acid sequence consisting of amino acid residues 20 to 471 of SEQID NO: 12 and a light chain consisting of an amino acid sequenceconsisting of amino acid residues 21 to 233 of SEQ ID NO: 13; anantibody consisting of a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 12and a light chain consisting of an amino acid sequence consisting ofamino acid residues 21 to 233 of SEQ ID NO: 14; an antibody consistingof a heavy chain consisting of an amino acid sequence consisting ofamino acid residues 20 to 471 of SEQ ID NO: 12 and a light chainconsisting of an amino acid sequence consisting of amino acid residues21 to 233 of SEQ ID NO: 15; and an antibody consisting of a heavy chainconsisting of an amino acid sequence consisting of amino acid residues20 to 471 of SEQ ID NO: 12 and a light chain consisting of an amino acidsequence consisting of amino acid residues 21 to 233 of SEQ ID NO: 16can be exemplified.

Furthermore, as an antibody which has another more preferred combinationof a heavy chain and a light chain described above, an antibodyconsisting of a heavy chain consisting of an amino acid sequence of SEQID NO: 9 and a light chain consisting of an amino acid sequence of SEQID NO: 13; an antibody consisting of a heavy chain consisting of anamino acid sequence of SEQ ID NO: 9 and a light chain consisting of anamino acid sequence of SEQ ID NO: 14; an antibody consisting of a heavychain consisting of an amino acid sequence of SEQ ID NO: 9 and a lightchain consisting of an amino acid sequence of SEQ ID NO: 15; an antibodyconsisting of a heavy chain consisting of an amino acid sequence of SEQID NO: 9 and a light chain consisting of an amino acid sequence of SEQID NO: 16; an antibody consisting of a heavy chain consisting of anamino acid sequence of SEQ ID NO: 9 and a light chain consisting of anamino acid sequence of SEQ ID NO: 17; an antibody consisting of a heavychain consisting of an amino acid sequence of SEQ ID NO: 9 and a lightchain consisting of an amino acid sequence of SEQ ID NO: 18; an antibodyconsisting of a heavy chain consisting of an amino acid sequence of SEQID NO: 9 and a light chain consisting of an amino acid sequence of SEQID NO: 19; an antibody consisting of a heavy chain consisting of anamino acid sequence of SEQ ID NO: 12 and a light chain consisting of anamino acid sequence of SEQ ID NO: 13; an antibody consisting of a heavychain consisting of an amino acid sequence of SEQ ID NO: 12 and a lightchain consisting of an amino acid sequence of SEQ ID NO: 14; an antibodyconsisting of a heavy chain consisting of an amino acid sequence of SEQID NO: 12 and a light chain consisting of an amino acid sequence of SEQID NO: 15; and an antibody consisting of a heavy chain consisting of anamino acid sequence of SEQ ID NO: 12 and a light chain consisting of anamino acid sequence of SEQ ID NO: 16 can be exemplified.

By combining a sequence having a high homology with the above-describedheavy chain amino acid sequence with a sequence having a high homologywith the above-described light chain amino acid sequence, it is possibleto select an antibody having a cytotoxic activity equivalent to that ofeach of the above-described antibodies. Such a homology is generally ahomology of 80% or more, preferably a homology of 90% or more, morepreferably a homology of 95% or more, most preferably a homology of 99%or more. Further, by combining an amino acid sequence wherein one toseveral amino acid residues are substituted, deleted or added in theheavy chain or light chain amino acid sequence, it is also possible toselect an antibody having a cytotoxic activity equivalent to that ofeach of the above-described antibodies.

The homology between two amino acid sequences can be determined usingdefault parameters of Blast algorithm version 2.2.2 (Altschul, StephenF., Thomas L. Madden, Alejandro A. SchAffer, Jinghui Zhang, Zheng Zhang,Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: anew generation of protein database search programs”, Nucleic Acids Res.25: 3389-3402). The Blast algorithm can be used also through theInternet by accessing the site www.ncbi.nlm.nih.gov/blast.

In the heavy chain amino acid sequence represented by SEQ ID NO: 9, 10,11 or 12 in the Sequence Listing, an amino acid sequence consisting ofamino acid residues 1 to 19 is a signal sequence, an amino acid sequenceconsisting of amino acid residues 20 to 141 is a variable region, and anamino acid sequence consisting of amino acid residues 142 to 471 is aconstant region. The sequence of SEQ ID NO: 9, 10, 11 and 12 are shownin FIGS. 3, 4, 5 and 6 respectively.

Further, in the light chain amino acid sequence represented by SEQ IDNO: 13, 14, 15, 16, 17, 18 or 19 in the Sequence Listing, an amino acidsequence consisting of amino acid residues 1 to 20 is a signal sequence,an amino acid sequence consisting of amino acid residues 21 to 128 is avariable region, and an amino acid sequence consisting of amino acidresidues 129 to 233 is a constant region. The sequence of SEQ ID NO: 13,14, 15, 16, 17, 18 and 19 are shown in FIGS. 7, 8, 9, 10, 11, 12 and 13respectively.

Further, the antibody of the invention includes a human antibody whichbinds to the same epitope as the M30 antibody. An anti-B7-H3 humanantibody refers to a human antibody having only a sequence of anantibody derived from a human chromosome. The anti-B7-H3 human antibodycan be obtained by a method using a human antibody-producing mousehaving a human chromosome fragment comprising heavy and light chaingenes of a human antibody (see Tomizuka, K. et al., Nature Genetics(1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26,pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic andApplied Aspects vol. 10, pp. 69-73 (Kitagawa, Y., Matuda, T. and Iijima,S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc.Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.).

Such a human antibody-producing mouse can be created specifically asfollows. A genetically modified animal in which endogenousimmunoglobulin heavy and light chain gene loci have been disrupted, andinstead, human immunoglobulin heavy and light chain gene loci have beenintroduced via a yeast artificial chromosome (YAC) vector or the like iscreated by producing a knockout animal and a transgenic animal andmating these animals.

Further, according to a recombinant DNA technique, by using cDNAsencoding each of such a heavy chain and a light chain of a humanantibody, and preferably a vector comprising such cDNAs, eukaryoticcells are transformed, and a transformant cell which produces arecombinant human monoclonal antibody is cultured, whereby the antibodycan also be obtained from the culture supernatant.

Here, as the host, for example, eukaryotic cells, preferably mammaliancells such as CHO cells, lymphocytes, or myeloma cells can be used.

Further, a method of obtaining a phage display-derived human antibodyselected from a human antibody library (see Wormstone, I. M. et al.,Investigative Ophthalmology & Visual Science. (2002) 43 (7), pp.2301-2308; Carmen, S. et al., Briefings in Functional Genomics andProteomics (2002), 1 (2), pp. 189-203; Siriwardena, D. et al.,Ophthalmology (2002) 109 (3), pp. 427-431, etc.) is also known.

For example, a phage display method in which a variable region of ahuman antibody is expressed on the surface of a phage as a single-chainantibody (scFv), and a phage which binds to an antigen is selected(Nature Biotechnology (2005), 23, (9), pp. 1105-1116) can be used.

By analyzing the gene of the phage selected based on the binding to anantigen, a DNA sequence encoding the variable region of a human antibodywhich binds to an antigen can be determined.

If the DNA sequence of scFv which binds to an antigen is determined, ahuman antibody can be obtained by preparing an expression vectorcomprising the sequence and introducing the vector into an appropriatehost to express it (WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236,WO 93/19172, WO 95/01438, WO 95/15388, Annu. Rev. Immunol. (1994) 12,pp. 433-455, Nature Biotechnology (2005) 23 (9), pp. 1105-1116).

If a newly produced human antibody binds to a partial peptide or apartial tertiary structure to which the M30 antibody binds, it can bedetermined that the human antibody binds to the same epitope as the M30antibody. Further, by confirming that the human antibody competes withthe M30 antibody for the binding to B7-H3 (that is, the human antibodyinhibits the binding between the M30 antibody and B7-H3), it can bedetermined that the human antibody binds to the same epitope as the M30antibody even if the specific epitope sequence or structure has not beendetermined. When it is confirmed that the human antibody binds to thesame epitope as the M30 antibody, the human antibody is stronglyexpected to have a cytotoxic activity equivalent to that of the M30antibody.

The chimeric antibodies, humanized antibodies, or human antibodiesobtained by the above-described method are evaluated for the bindingproperty to an antigen by a known method or the like, and a preferredantibody can be selected.

As one example of another index for use in the comparison of theproperties of antibodies, the stability of antibodies can beexemplified. The differential scanning calorimetry (DSC) is a devicecapable of quickly and accurately measuring a thermal denaturationmidpoint temperature (Tm) to be used as a favorable index of therelative conformational stability of proteins. By measuring the Tmvalues using DSC and comparing the values, a difference in thermalstability can be compared. It is known that the storage stability ofantibodies shows some correlation with the thermal stability ofantibodies (Lori Burton, et. al., Pharmaceutical Development andTechnology (2007) 12, pp. 265-273), and a preferred antibody can beselected by using thermal stability as an index. Examples of otherindices for selecting antibodies include the following features: theyield in an appropriate host cell is high; and the aggregability in anaqueous solution is low. For example, an antibody which shows thehighest yield does not always show the highest thermal stability, andtherefore, it is necessary to select an antibody most suitable for theadministration to humans by making comprehensive evaluation based on theabove-described indices.

In the invention, a modified variant of the antibody is also included.The modified variant refers to a variant obtained by subjecting theantibody of the invention to chemical or biological modification.Examples of the chemically modified variant include variants chemicallymodified by linking a chemical moiety to an amino acid skeleton,variants chemically modified with an N-linked or O-linked carbohydratechain, etc. Examples of the biologically modified variant includevariants obtained by post-translational modification (such as N-linkedor O-linked glycosylation, N- or C-terminal processing, deamidation,isomerization of aspartic acid, or oxidation of methionine), andvariants in which a methionine residue has been added to the N terminusby being expressed in a prokaryotic host cell.

Further, an antibody labeled so as to enable the detection or isolationof the antibody or an antigen of the invention, for example, anenzyme-labeled antibody, a fluorescence-labeled antibody, and anaffinity-labeled antibody are also included in the meaning of themodified variant. Such a modified variant of the antibody of theinvention is useful for improving the stability and blood retention ofthe original antibody of the invention, reducing the antigenicitythereof, detecting or isolating such an antibody or an antigen, and soon.

Further, by regulating the modification of a glycan which is linked tothe antibody of the invention (glycosylation, defucosylation, etc.), itis possible to enhance an antibody-dependent cellular cytotoxicactivity. As the technique for regulating the modification of a glycanof antibodies, WO 99/54342, WO 00/61739, WO 02/31140, etc. are known.However, the technique is not limited thereto. In the antibody of theinvention, an antibody in which the modification of a glycan isregulated is also included.

In the case where an antibody is produced by first isolating an antibodygene and then introducing the gene into an appropriate host, acombination of an appropriate host and an appropriate expression vectorcan be used. Specific examples of the antibody gene include acombination of a gene encoding a heavy chain sequence of an antibodydescribed in this specification and a gene encoding a light chainsequence thereof. When a host cell is transformed, it is possible toinsert the heavy chain sequence gene and the light chain sequence geneinto the same expression vector, and also into different expressionvectors separately.

In the case where eukaryotic cells are used as the host, animal cells,plant cells, and eukaryotic microorganisms can be used. As the animalcells, mammalian cells, for example, simian COS cells (Gluzman, Y.,Cell, (1981) 23, pp. 175-182, ATCC CRL-1650), murine fibroblasts NIH3T3(ATCC No. CRL-1658), and dihydrofolate reductase-deficient strains(Urlaub, G. and Chasin, L. A., Proc. Natl. Acad. Sci. USA (1980) 77, pp.4126-4220) of Chinese hamster ovarian cells (CHO cells; ATCC: CCL-61)can be exemplified.

In the case where prokaryotic cells are used, for example, Escherichiacoli and Bacillus subtilis can be exemplified.

By introducing a desired antibody gene into these cells throughtransformation, and culturing the thus transformed cells in vitro, theantibody can be obtained. In the above-described culture method, theyield may sometimes vary depending on the sequence of the antibody, andtherefore, it is possible to select an antibody which is easily producedas a pharmaceutical by using the yield as an index among the antibodieshaving an equivalent binding activity. Therefore, in the antibody of theinvention, an antibody obtained by a method of producing an antibody,characterized by including a step of culturing the transformed host celland a step of collecting a desired antibody from a cultured productobtained in the culturing step is also included.

It is known that a lysine residue at the carboxyl terminus of the heavychain of an antibody produced in a cultured mammalian cell is deleted(Journal of Chromatography A, 705: 129-134 (1995)), and it is also knownthat two amino acid residues (glycine and lysine) at the carboxylterminus of the heavy chain of an antibody produced in a culturedmammalian cell are deleted and a proline residue newly located at thecarboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83(2007)). However, such deletion and modification of the heavy chainsequence do not affect the antigen-binding affinity and the effectorfunction (the activation of a complement, the antibody-dependentcellular cytotoxicity, etc.) of the antibody. Therefore, in theinvention, an antibody subjected to such modification is also included,and a deletion variant in which one or two amino acids have been deletedat the carboxyl terminus of the heavy chain, a variant obtained byamidation of the deletion variant (for example, a heavy chain in whichthe carboxyl terminal proline residue has been amidated), and the likecan be exemplified. The type of deletion variant having a deletion atthe carboxyl terminus of the heavy chain of the antibody according tothe invention is not limited to the above variants as long as theantigen-binding affinity and the effector function are conserved. Thetwo heavy chains constituting the antibody according to the inventionmay be of one type selected from the group consisting of a full-lengthheavy chain and the above-described deletion variant, or may be of twotypes in combination selected therefrom. The ratio of the amount of eachdeletion variant can be affected by the type of cultured mammalian cellswhich produce the antibody according to the invention and the cultureconditions, however, a case where one amino acid residue at the carboxylterminus has been deleted in both of the two heavy chains contained asmain components in the antibody according to the invention can beexemplified.

As isotype of the antibody of the invention, for example, IgG (IgG1,IgG2, IgG3, IgG4) can be exemplified, and IgG1 or IgG2 can beexemplified preferably.

As the function of the antibody, generally an antigen-binding activity,an activity of neutralizing the activity of an antigen, an activity ofenhancing the activity of an antigen, an antibody-dependent cellularcytotoxicity (ADCC) activity and a complement-dependent cytotoxicity(CDC) activity can be exemplified. The function of the antibody of theinvention is a binding activity to B7-H3, preferably anantibody-dependent cell-mediated phagocytosis (ADCP) activity, morepreferably a cytotoxicity activity (antitumor activity) to tumor cellmediated by an ADCP activity. Further, the antibody of the invention mayhave an ADCC activity and/or a CDC activity in addition to an ADCPactivity.

The obtained antibody can be purified to homogeneity. The separation andpurification of the antibody may be performed employing a conventionalprotein separation and purification method. For example, the antibodycan be separated and purified by appropriately selecting and combiningcolumn chromatography, filter filtration, ultrafiltration, saltprecipitation, dialysis, preparative polyacrylamide gel electrophoresis,isoelectric focusing electrophoresis, and the like (Strategies forProtein Purification and Characterization: A Laboratory Course Manual,Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press(1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, ColdSpring Harbor Laboratory (1988)), but the method is not limited thereto.

Examples of such chromatography include affinity chromatography, ionexchange chromatography, hydrophobic chromatography, gel filtrationchromatography, reverse phase chromatography, and adsorptionchromatography.

Such chromatography can be performed employing liquid chromatographysuch as HPLC or FPLC.

As a column to be used in affinity chromatography, a Protein A columnand a Protein G column can be exemplified. For example, as a columnusing a Protein A column, Hyper D, POROS, Sepharose FF (Pharmacia) andthe like can be exemplified.

Further, by using a carrier having an antigen immobilized thereon, theantibody can also be purified utilizing the binding property of theantibody to the antigen.

[Antitumor Compound]

The antitumor compound to be conjugated to the antibody-drug conjugateof the present invention is explained. The antitumor compound is notparticularly limited if it is a compound having an antitumor effect anda substituent group or a partial structure allowing connecting to alinker structure. When a part or whole linker is cleaved in tumor cells,the antitumor compound moiety is released to exhibit the antitumoreffect of the antitumor compound. As the linker is cleaved at aconnecting position to drug, the antitumor compound is released in itsintrinsic structure to exhibit its intrinsic antitumor effect.

Examples of the antitumor compound can include doxorubicin,daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine,vinblastine, methotrexate, platinum-based antitumor agent (cisplatin orderivatives thereof), taxol or derivatives thereof, and camptothecin orderivatives thereof (antitumor agent described in Japanese PatentLaid-Open No. 6-87746). In the antibody-drug conjugate of the presentinvention, exatecan as a camptothecin derivative(((1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione;shown in the following formula) can be preferably used.

Although having an excellent antitumor effect, exatecan has not beencommercialized as an antitumor drug. The compound can be easily obtainedby a known method and the amino group at position 1 can be preferablyused as a connecting position to the linker structure. Further, althoughexatecan can be also released in tumor cells while part of the linker isstill attached thereto, it is an excellent compound exhibiting anexcellent antitumor effect even in such case.

With regard to the antibody-drug conjugate, the number of conjugateddrug molecules per antibody molecule is a key factor having an influenceon the efficacy and safety. Production of the antibody-drug conjugate isperformed by defining the reaction condition including the amounts ofuse of raw materials and reagents for reaction so as to have a constantnumber of conjugated drug molecules, a mixture containing differentnumbers of conjugated drug molecules is generally obtained unlike thechemical reaction of a low-molecular-weight compound. The number ofdrugs conjugated in an antibody molecule is expressed or specified bythe average value, that is, the average number of conjugated drugmolecules. Unless specifically described otherwise as a principle, thenumber of conjugated drug molecules means an average value except in acase in which it represents an antibody-drug conjugate having a specificnumber of conjugated drug molecules that is included in an antibody-drugconjugate mixture having different number of conjugated drug molecules.The number of exatecan molecules conjugated to an antibody molecule iscontrollable, and as an average number of conjugated drug molecules perantibody, about 1 to 10 exatecans can be bound. Preferably, it is 2 to8, and more preferably 3 to 8. Meanwhile, a person skilled in the artcan design a reaction for conjugating a required number of drugmolecules to an antibody molecule based on the description of theExamples of the present application and can obtain an antibodyconjugated with a controlled number of exatecan molecules.

Because exatecan has a camptothecin structure, it is known that theequilibrium shifts to a structure with a closed lactone ring (closedring) in an aqueous acidic medium (for example, pH 3 or so) but itshifts to a structure with an open lactone ring (open ring) in anaqueous basic medium (for example, pH 10 or so). A drug conjugate beingintroduced with an exatecan residue corresponding to the closed ringstructure and the open ring structure is also expected to have the sameantitumor effect and it is needless to say that any of them is withinthe scope of the present invention.

[Linker Structure]

With regard to the antibody-drug conjugate of the present invention, thelinker structure for conjugating an antitumor drug to the antibody isexplained. The linker has a structure of the following structure:

-L¹-L²-L^(P)-NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)-.

The antibody is connected to the terminal of L¹ (terminal opposite tothe connection to L²), and the antitumor drug is connected to theterminal of L^(c) (terminal opposite to the connection to L^(b)).

n¹ represents an integer of 0 to 6 and is preferably an integer of 1 to5, and more preferably 1 to 3.

1. L¹

L¹ is a moiety in the linker represented by the following structure:

-   -(Succinimid-3-yl-N)—(CH₂)n²-C(═O)—,-   —CH₂—C(═O)—NH—(CH₂)n³-C(═O)—,-   —C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)-, or-   —C(═O)—(CH₂) n⁴-C(═O)—    In the above, n² is an integer of 2 to 8, n³ is an integer of 1 to    8, and n⁴ is an integer of 1 to 8.

In the linker having a structure represented by-(Succinimid-3-yl-N)—(CH₂)n²-C(═O)— of L¹, “-(Succinimid-3-yl-N)—” has astructure represented by the following formula:

Position 3 of the above partial structure is a connecting position tothe antibody. The bond to the antibody at position 3 is characterized bybonding with thioether formation. On the other hand, the nitrogen atomat position 1 of the structure moiety is connected to the carbon atom ofmethylene which is present within the linker including the structure.Specifically, -(Succinimid-3-yl-N)—(CH₂) n²-C(═O)-L²- is a structurerepresented by the following formula (herein, “antibody-S—” originatesfrom an antibody).

In the formula, n² is an integer of 2 to 8, and preferably 2 to 5.

In the linker having a structure represented by—CH₂—C(═O)—NH—(CH₂)n³-C(═O)— of L¹, n³ is an integer of 1 to 8,preferably 2 to 6. This linker is connected to the antibody at itscarbon atom of terminal methylene and has the following structure forconnecting by thioether formation, as with the preceding linker (herein,“antibody-S—” originates from an antibody).Antibody-S—CH₂—C(═O)—NH—(CH₂)n³-C(═O)-L²-.

In the linker having a structure represented by—C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)- of L¹,“—(N-ly-3-diminiccuS)-” has a structure represented by the followingformula:

In this structure moiety, the nitrogen atom at position 1 is connectedto the carbon atom of methylene present in the linker structurecontaining this structure. The carbon atom at position 3 is connected tothe terminal sulfur atom of —S—(CH₂)n⁶-C(═O)— of L² in the linker. Thismoiety —S—(CH₂)n⁶-C(═O)— of L² in the linker forms a combined linkerstructure only with —C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)- of L¹in the linker. In the above, “-cyc.Hex(1,4)-” contained in the linkerrepresents a 1,4-cyclohexylene group. In the linker,—C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)- is connected to theantibody with amide bond formation at its terminal carbonyl carbon(herein, “antibody-NH—” originates from an antibody).

The amino group of the antibody for this amide bond formation is theterminal amino group of a side chain of a lysine residue in the antibodyor an amino group at the N terminal of the antibody. Said linker of astructure can connect by forming ester bond with the hydroxy group of anamino acid in the antibody other than such amide bond.

The structure moiety “-cyc.Hex(1,4)-” contained in said linker may be adivalent saturated cyclic alkylene group other than the1,4-cyclohexylene group, i.e., a divalent cyclic saturated hydrocarbongroup such as a cyclobutylene group, a cyclopentylene group, acycloheptalene group, or a cyclooctalene group, a divalent aromatichydrocarbon group such as a phenylene group or a naphthylene group, or a5- or 6-membered saturated, partially saturated, or aromatic divalentheterocyclic group containing 1 or 2 heteroatoms. Alternatively, thismoiety may be a divalent alkylene group having 1 to 4 carbon atoms. Theconnection to the divalent group may occur at adjacent positions or atdistant positions.

In the linker having a structure represented by —C(═O)—(CH₂)n⁴-C(═O)— asTi, n⁴ is an integer of 1 to 8, and preferably 2 to 6. This linker isalso connected by amide bond formation at its terminal carbonyl groupwith an amino group of the antibody, as with the linkers mentioned above(see the following formula; in the structure thereof, “antibody-NH—”originates from an antibody).

Antibody-NH—C(═O)—(CH₂)n⁴-C(═O)-L²-.

Specific examples of L¹ in the linker can include

-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—-(Succinimid-3-yl-N)—CH₂CH₂CH₂—C(═O)—-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂—C(═O)—-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)——CH₂C(═O)NH—CH₂—C(═O)—,—CH₂C(═O)NH—CH₂CH₂—C(═O)——CH₂C(═O)NH—CH₂CH₂CH₂—C(═O)——CH₂C(═O)NH—CH₂CH₂CH₂CH₂—C(═O)——CH₂C(═O)NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—

—C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)-—C(═O)-Aryl-CH₂—(N-ly-3-diminiccuS)-—C(═O)-cyc.Het-CH₂—(N-ly-3-diminiccuS)-

—C(═O)—CH₂CH₂—C(═O)—

-   C(═O)—CH₂CH₂CH₂—C(═O)—-   C(═O)—CH₂CH₂CH₂CH₂—C(═O)—-   C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)—-   C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)—.-   (Aryl represents a divalent aromatic hydrocarbon group, and cyc.Het    represents a divalent cyclic heterocyclic group).

2. L²

L² is a linker represented by the following structure:

-   —NH—(CH₂CH₂O)n⁵-CH₂—CH₂—C(═O)—, or-   —S—(CH₂)n⁶-C(═O)—,    L² may not be present, and in such a case, L² is a single bond. In    the above, n⁵ is an integer of 1 to 6, and n⁶ is an integer of 1 to    6.

In the linker having a structure of —NH—(CH₂CH₂O)n⁵-CH₂—CH₂—C(═O)— asL², n⁵ is an integer of 1 to 6, and preferably 2 to 4. This moiety inthe linker is connected to L¹ at its terminal amino group and isconnected to L^(P) at its carbonyl group at the other terminal.

In the linker having a structure of —S—(CH₂)n⁶-C(═O)— as L2, n⁶ is aninteger of 1 to 6, and preferably 2 to 4.

Specific examples of L² can include

-   —NH—CH₂CH₂O—CH₂CH₂—C(═O)—,-   —NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)—,-   —NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)—,-   —NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)—,-   —NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)—,-   —NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)—.

When L² is —S—(CH₂)n⁶-C(═O)—, L¹ to be combined therewith is—C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)-. Specific examples of-L¹-L²- can include

-   —C(═O)-cyc.Hex (1,4)-CH₂— (N-ly-3-diminiccuS)-S—CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—(N-ly-3-diminiccuS)-S—CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—(N-ly-3-diminiccuS)-S—CH₂CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—(N-ly-3-diminiccuS)-S—CH₂CH₂CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex (1,4)-CH₂—(N-ly-3-diminiccuS)-S—CH₂CH₂CH₂CH₂CH₂—    C(═O)—-   —C(═O)-cyc.Hex    (1,4)-CH₂—(N-ly-3-diminiccuS)-S—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)—.

3. L^(P)

The linker L^(P) is a peptide residue consisting of 2 to 7 amino acids.Specifically, it consists of an oligopeptide residue in which 2 to 6amino acids are linked by a peptide bonding. The linker L^(P) isconnected to L² at its N terminal and is connected to the amino group of—NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)-moiety of the linker at its C terminal.The amino acid constituting L^(P) in the linker is not particularlylimited, however, examples thereof include an L- or a D-amino acid,preferably an L-amino acid. And, it can be an amino acid having astructure such as β-alanine, ε-aminocaproic acid, or γ-aminobutyric acidin addition to an α-amino acid, further, it can be a non-natural typeamino acid such as N-methylated amino acid.

The amino acid sequence of L^(P) is not particularly limited, butexamples of the constituting amino acid include phenylalanine (Phe; F),tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A),valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S),glutamic acid (Glu; E), and aspartic acid (Asp; D). Among them,preferred examples include phenylalanine, glycine, valine, lysine,citrulline, serine, glutamic acid, and aspartic acid. Depending on thetype of the amino acid, drug release pattern can be controlled. Thenumber of the amino acid can be between 2 to 7.

Specific examples of L^(P) can include

-   -GGF--   -DGGF--   -(D-)D-GGF--   -EGGF--   -GGFG--   -SGGF--   -KGGF--   -DGGFG--   -GGFGG--   -DDGGFG--   -KDGGFG--   -GGFGGGF-    [in the above, “(D-)D” represents a D-aspartic acid]. Particularly    preferred examples of L^(P) for the antibody-drug conjugate of the    present invention can include -GGFG-.

In the structure represented by —NH—(CH₂)n¹- within the linker, n¹ is aninteger of 0 to 6 and is preferably an integer of 1 to 5, and morepreferably 1 to 3. The amino group of this moiety is connected to the Cterminal of L^(P) in the linker.

4. L^(a)

The linker L^(a) is represented by any of structures —C(═O)—NH—,—NR¹—(CH₂)n⁷-, and —O— or is a single bond.

In the above, n⁷ is an integer of 1 to 6, R¹ is a hydrogen atom, analkyl group having 1 to 6 carbon atoms, —(CH₂)n⁸-COOH, or —(CH₂)n⁹-OH,n⁸ is an integer of 1 to 4, and n⁹ is an integer of 1 to 6.

The amide structure —C(═O)—NH— within linker L^(a) is connected to L^(b)at its nitrogen atom side. In the structure moiety of —NR¹—(CH₂)n⁷-within L^(a), n⁷ is an integer of 1 to 6, and preferably 1 to 3. Thismoiety is connected to L^(b) at its methylene side. R¹ is a hydrogenatom or an alkyl group having 1 to 6 carbon atoms. The alkyl grouphaving 1 to 6 carbon atoms may be linear or branched. Examples thereofcan include a methyl group, an ethyl group, a propyl group, an isopropylgroup, a butyl group, an isobutyl group, a sec-butyl group, a tert-butylgroup, a pentyl group, an isopentyl group, a 2-methylbutyl group, aneopentyl group, a 1-ethylpropyl group, a hexyl group, an isohexylgroup, a 4-methylpentyl group, a 3-methylpentyl group, a 2-methylpentylgroup, a 1-methylpentyl group, a 3,3-dimethylbutyl group, a2,2-dimethylbutyl group, a 1,1-dimethylbutyl group, a 1,2-dimethylbutylgroup, a 1,3-dimethylbutyl group, a 2,3-dimethylbutyl group, and a2-ethylbutyl group. Of them, a methyl group or an ethyl group ispreferred. When R¹ has a structure represented by —(CH₂)n⁸-COOH, n⁸ isan integer of 1 to 4, and preferably 1 or 2. When R¹ has a structurerepresented by —(CH₂)n⁹-OH, n⁹ is an integer of 1 to 6, and preferably 1or 2. R¹ is preferably a hydrogen atom, a methyl group, an ethyl group,—CH₂COOH, —CH₂CH₂—COOH, or —CH₂CH₂—OH, and more preferably a hydrogenatom, a methyl group, or —CH₂COOH. It is further preferably a hydrogenatom. The L^(a) moiety of the linker may be —O— or a single bond.

5. L^(b)

The linker L^(b) is any of structures —CR²(—R³)—, —O—, and —NR⁴— or is asingle bond. In the above, R² and R³ each independently represents ahydrogen atom, an alkyl group having 1 to 6 carbon atoms,—(CH₂)n^(a)-NH₂, (CH₂)n^(b)-COOH, or —(CH₂)n^(c)-OH, R⁴ is a hydrogenatom or an alkyl group having 1 to 6 carbon atoms, n^(a) is an integerof 0 to 6, n^(b) is an integer of 1 to 4, and n^(c is an integer of) 0to 4. When n^(a) or n^(c) is 0, R² and R³ are not the same each other.

When each of R² and R³ is an alkyl group, this alkyl group isinterpreted as defined in the alkyl group of R¹. When R² and R³ has astructure of —(CH₂)n^(a)-NH₂, n^(a) is an integer of 0 to 6, andpreferably 0, or is 3 to 5. When n^(a) is 0, R² and R³ are not the sameas each other. When R² and R³ has a structure of —(CH₂)n^(b)-COOH, n^(b)is an integer of 1 to 4, and preferably 1 or 2. When R² and R³ has astructure of —(CH₂)n^(c)-OH, n^(c) is an integer of 0 to 4, andpreferably 1 or 2.

Each of R² and R³ is preferably a hydrogen atom, a methyl group, anethyl group, —NH₂, —CH₂CH₂CH₂NH₂, CH₂CH₂CH₂CH₂NH₂,—CH₂CH₂CH₂CH₂CH₂CH₂NH₂, —CH₂COOH, —CH₂CH₂—COOH, —CH₂OH, or —CH₂CH₂—OH,and more preferably a hydrogen atom, a methyl group, —NH₂,—CH₂CH₂CH₂CH₂NH₂, —CH₂COOH, —CH₂CH₂—COOH, —CH₂OH, or —CH₂CH₂—OH. Theyare further preferably hydrogen atoms.

When R⁴ is an alkyl group having 1 to 6 carbon atoms, this alkyl groupis interpreted as defined in the alkyl group of R¹. R⁴ is preferably ahydrogen atom or a methyl group, and more preferably a hydrogen atom.

Specific examples of the structure represented by

-   —NH—(CH₂)n¹-L^(a)-L^(b)- as the linker can include-   —NH—CH₂—-   NH—CH(-Me)--   NH—C(-Me)₂--   NH—CH₂— CHMe --   NH—CH(—CH₂OH)—-   NH—CH(—CH₂COOH)—-   NH—CH(—CH₂CH₂COOH)—-   NH—CH(—CH₂CH₂CH₂CH₂NH₂)—-   —NH—CH₂CH₂—-   NH—CH₂—O—CH₂—-   NH—CH₂CH₂— O—-   NH—CH₂CH₂—O—CH₂—-   NH—CH₂CH₂C(-Me)₂--   NH—CH₂CH₂NH—-   NH—CH₂CH₂NH—CH₂—-   NH—CH₂CH₂NMe-CH₂—-   —NH—CH₂CH₂NH—CH₂CH₂—-   —NH—CH₂CH₂NMe-CH₂CH₂—-   —NH—CH₂CH₂N(—CH₂COOH)—CH₂—-   —NH—CH₂CH₂N(—CH₂CH₂OH)—CH₂—-   —NH—CH₂CH₂N(—CH₂CH₂OH)—CH₂CH₂—-   —NH—CH₂CH₂CH₂C(═O)—NHCH(—CH₂OH)—-   —NH—CH₂CH₂CH₂C(═O)—NHCH(—CH₂COOH)—-   —NH—CH₂CH₂CH₂C(═O)—NHCH(—CH₂CH₂CH₂CH₂NH₂)—-   —NH—CH₂CH₂CH₂—-   —NH—CH₂CH₂CH₂CH₂—-   —NH—CH₂CH₂CH₂CH₂CH₂—-   —NH—CH₂CH₂CH₂CH₂CH(NH₂)—.

Of them, preferred examples thereof can include

-   —NH—CH₂—-   —NH—CH₂— CH (Me) --   —NH—CH(—CH₂OH)—-   —NH—CH(—CH₂CH₂COOH)—-   —NH—CH₂CH₂—-   —NH—CH₂— O—CH₂—-   —NH—CH₂CH₂—O—-   —NH—CH₂CH₂—O—CH₂—-   —NH—CH₂CH₂C(-Me)₂--   —NH—CH₂CH₂NH—-   —NH—CH₂CH₂NH—CH₂—-   —NH—CH₂CH₂NMe-CH₂—-   —NH—CH₂CH₂NMe-CH₂CH₂—-   —NH—CH₂CH₂N(—CH₂COOH)—CH₂—-   —NH—CH₂CH₂N(—CH₂CH₂OH)—CH₂—-   —NH—CH₂CH₂N(—CH₂CH₂OH)—CH₂CH₂—-   —NH—CH₂CH₂CH₂C(═O)—NHCH(—CH₂OH)—-   —NH—CH₂CH₂CH₂C(═O)—NHCH(—CH₂COOH)—-   —NH—CH₂CH₂CH₂—-   —NH—CH₂CH₂CH₂CH₂—-   —NH—CH₂CH₂CH₂CH₂CH₂—.

More preferred examples thereof can include

-   —NH—CH₂—-   —NH—CH₂CH₂—-   —NH—CH₂—O—CH₂—-   —NH—CH₂CH₂—O—-   —NH—CH₂CH₂—O—CH₂—-   —NH—CH₂CH₂NH—-   —NH—CH₂CH₂NH—CH₂—-   —NH—CH₂CH₂N(—CH₂COOH)—CH₂—-   —NH—CH₂CH₂N(—CH₂CH₂OH)—CH₂CH₂—-   —NH—CH₂CH₂CH₂C(═O)—NHCH(—CH₂COOH)—-   —NH—CH₂CH₂CH₂—-   —NH—CH₂CH₂CH₂CH₂—-   —NH—CH₂CH₂CH₂CH₂CH₂—.

Further preferred examples thereof can include

-   —NH—(CH₂)₃—,-   —NH—CH₂—O—CH₂—, and —NH—(CH₂)₂—O—CH₂—.

6. L^(c)

The linker L^(c) is —CH₂— or —C(═O)—. Said linker is connected to theantitumor compound. L^(c) of the linker is more preferably —C(═O)—.

In the linker, the chain length of —NH—(CH₂)n¹-L^(a)-L^(b)-L^(c) ispreferably a chain length of 4 to 7 atoms, and more preferably a chainlength of 5 or 6 atoms.

With regard to the antibody-drug conjugate of the present invention,when it is transferred to the inside of tumor cells, the linker moietyis cleaved and the drug derivative having a structure represented byNH₂—(CH₂)n¹-L^(a)-L^(b)-L^(c)-(NH-DX) is released to express anantitumor action. Examples of the antitumor derivative exhibiting anantitumor effect by releasing from the antibody-drug conjugate of thepresent invention include an antitumor derivative having a structuremoiety in which the structure represented by —NH—(CH₂)n¹-L^(a)-L^(b)- ofthe linker is bound with L^(c) and has a terminal amino group, and theparticularly preferred include the followings.

-   NH₂—CH₂CH₂—C(═O)—(NH-DX)-   NH₂—CH₂CH₂CH₂—C(═O)—(NH-DX)-   NH₂—CH₂—O—CH₂—C(═O)—(NH-DX)-   NH₂—CHCH₂—O—CH₂—C(═O)—(NH-DX)

Meanwhile, in case of NH₂—CH₂—O—CH₂—C(═O)—(NH-DX), it was confirmedthat, as the aminal structure in the molecule is unstable, it againundergoes a self-degradation to release the followingHO—CH₂—C(═O)—(NH-DX). Those compounds can be also preferably used as aproduction intermediate of the antibody-drug conjugate of the presentinvention.

For the antibody-drug conjugate of the present invention in whichexatecan is used as a drug, it is preferable that the drug-linkerstructure moiety having the following structure[-L¹-L²-L^(P)-NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)-(NH-DX)] is connected to anantibody. The average conjugated number of the drug-linker structuremoiety per antibody can be 1 to 10. Preferably, it is 2 to 8, and morepreferably 3 to 8.

-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   —C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   —C(═O)-cyc.Hex    (1,4)-CH₂—(N-ly-3-diminiccuS)-S—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)

Among them, the more preferred are the followings.

-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   —C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   —C(═O)-cyc.Hex    (1,4)-CH₂—(N-ly-3-diminiccuS)-S—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX).

The particularly preferred are the followings.

-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX).

With regard to the linker structure for conjugating the antibody and adrug in the antibody-drug conjugate of the present application, thepreferred linker can be constructed by connecting preferred structuresshown for each part of the linker explained above. As for the linkerstructure, those with the following structure can be preferably used.Meanwhile, the left terminal of the structure is a connecting positionwith the antibody and the right terminal is a connecting position withthe drug.

-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex    (1,4)-CH₂—(N-ly-3-diminiccuS)-S—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—

Among them, the more preferred are the followings.

-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—-   —C(═O)-cyc.Hex    (1,4)-CH₂—(N-ly-3-diminiccuS)-S—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—.

The particularly preferred include the followings.

-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—-   -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—.

[Production Method]

Next, explanations are given for the representative method for producingthe antibody-drug conjugate of the present invention or a productionintermediate thereof. Meanwhile, the compounds are hereinbelow describedwith the compound number shown in each reaction formula. Specifically,they are referred to as a “compound of the formula (1)”, a “compound(1)”, or the like. The compounds with numbers other than those are alsodescribed similarly.

1. Production Method 1

The antibody-drug conjugate represented by the formula (1) in which theantibody is bound to the linker structure via thioether can be producedby the following method, for example.

[In the formula, AB represents an antibody with a sulfhydryl group, andL^(1′) represents L¹ linker structure in which the linker terminal is amaleimidyl group (formula shown below)

(in the formula, the nitrogen atom is the connecting position)or the terminal is halogen, and represents a group in which the-(Succinimid-3-yl-N)— moiety in -(Succinimid-3-yl-N)—(CH₂)n²-C(═O)— ofL¹ is a maleimidyl group or a halogen-CH₂C(═O) NH—(CH₂) n³-C(═O)— groupin which terminal methylene in —CH₂C(═O)NH—(CH₂)n³-C(═O)— of L¹ ishalogenated to form haloacetamide. Further, the —(NH-DX) represents astructure represented by the following formula:

and it represents a group that is derived by removing one hydrogen atomof the amino group at position 1 of exatecan. Further, the compound ofthe formula (1) in the above reaction formula is described as astructure in which one structure moiety from drug to the linker terminalconnects to one antibody. However, it is only the description given forthe sake of convenience, and there are actually many cases in which aplurality of the structure moieties are connected to one antibodymolecule. The same applies to the explanation of the production methoddescribed below.]

Specifically, the antibody-drug conjugate (1) can be produced byreacting the compound (2), which is obtainable by the method describedbelow, with the antibody (3a) having a sulfhydryl group.

The antibody (3a) having a sulfhydryl group can be obtained by a methodwell known in the art (Hermanson, G. T, Bioconjugate Techniques, pp.56-136, pp. 456-493, Academic Press (1996)). Examples include: Traut'sreagent is reacted with the amino group of the antibody; N-succinimidylS-acetylthioalkanoates are reacted with the amino group of the antibodyfollowed by reaction with hydroxylamine; after reacting withN-succinimidyl 3-(pyridyldithio)propionate, the antibody is reacted witha reducing agent; the antibody is reacted with a reducing agent such asdithiothreitol, 2-mercaptoethanol, and tris(2-carboxyethyl)phosphinehydrochloride (TCEP) to reduce the disulfide bond in a hinge part in theantibody to form a sulfhydryl group, but it is not limited thereto.

Specifically, using 0.3 to 3 molar equivalents of TCEP as a reducingagent per disulfide in hinge part in the antibody and reacting with theantibody in a buffer solution containing a chelating agent, the antibodywith partially or completely reduced disulfide in hinge part in theantibody can be obtained. Examples of the chelating agent includeethylenediamine tetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). It can be used at concentration of 1 mM to 20mM. Examples of the buffer solution which may be used include a solutionof sodium phosphate, sodium borate, or sodium acetate. As a specificexample, by reacting the antibody with TCEP at 4° C. to 37° C. for 1 to4 hours, the antibody (3a) having partially or completely reducedsulfhydryl group can be obtained.

Meanwhile, by performing the reaction for adding a sulfhydryl group to adrug-linker moiety, the drug-linker moiety can be conjugated by athioether bond.

Next, using 2 to 20 molar equivalents of the compound (2) per theantibody (3a) having a sulfhydryl group, the antibody-drug conjugate (1)in which 2 to 8 drug molecules are conjugated per antibody can beproduced. Specifically, it is sufficient that the solution containingthe compound (2) dissolved therein is added to a buffer solutioncontaining the antibody (3a) having a sulfhydryl group for the reaction.Herein, examples of the buffer solution which may be used include sodiumacetate solution, sodium phosphate, and sodium borate. pH for thereaction is 5 to 9, and more preferably the reaction is performed nearpH 7. Examples of the solvent for dissolving the compound (2) include anorganic solvent such as dimethyl sulfoxide (DMSO), dimethylformamide(DMF), dimethyl acetamide (DMA), and N-methyl-2-pyridone (NMP). It issufficient that the organic solvent solution containing the compound (2)dissolved therein is added at 1 to 20% v/v to a buffer solutioncontaining the antibody (3a) having a sulfhydryl group for the reaction.The reaction temperature is 0 to 37° C., more preferably 10 to 25° C.,and the reaction time is 0.5 to 2 hours. The reaction can be terminatedby deactivating the reactivity of unreacted compound (2) with athiol-containing reagent. Examples of the thiol-containing reagentinclude cysteine and N-acetyl-L-cysteine (NAC). More specifically, 1 to2 molar equivalents of NAC are added to the compound (2) used and, byincubating at room temperature for 10 to 30 minutes, the reaction can beterminated.

The produced antibody-drug conjugate (1) can be subjected to, afterconcentration, buffer exchange, purification, and measurement ofantibody concentration and average number of conjugated drug moleculesper antibody molecule according to common procedures described below,identification of the antibody-drug conjugate (1).

Common Procedure A: Concentration of Aqueous Solution of Antibody orAntibody-Drug Conjugate

To a Amicon Ultra (50,000 MWCO, Millipore Corporation) container, asolution of antibody or antibody-drug conjugate was added and thesolution of the antibody or antibody-drug conjugate was concentrated bycentrifugation (centrifuge for 5 to 20 minutes at 2000 G to 3800 G)using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.).

Common Procedure B: Measurement of Antibody Concentration

Using a UV detector (Nanodrop 1000, Thermo Fisher Scientific Inc.),measurement of the antibody concentration was performed according to themethod defined by the manufacturer. At that time, 280 nm absorptioncoefficient different for each antibody was used (1.3 mLmg⁻¹ cm⁻¹ to 1.8mLmg⁻¹ cm⁻¹).

Common procedure C-1: Buffer Exchange for antibody

NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation)using Sephadex G-25 carrier was equilibrated with phosphate buffer (10mM, pH 6.0) (it is referred to as PBS6.0/EDTA in the specification)containing sodium chloride (137 mM) and ethylene diamine tetraaceticacid (EDTA, 5 mM) according to the method defined by the manufacturer'sinstruction manual. Aqueous solution of the antibody was applied in anamount of 2.5 mL to single NAP-25 column, and then the fraction (3.5 mL)eluted with 3.5 mL of PBS6.0/EDTA was collected. The resulting fractionwas concentrated by the Common procedure A. After measuring theconcentration of the antibody using the Common procedure B, the antibodyconcentration was adjusted to 10 mg/mL using PBS6.0/EDTA.

Common Procedure C-2: Buffer Exchange for Antibody

NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation)using Sephadex G-25 carrier was equilibrated with phosphate buffer (50mM, pH 6.5) (it is referred to as PBS6.5/EDTA in the specification)containing sodium chloride (50 mM) and EDTA (2 mM) according to themethod defined by the manufacturer. Aqueous solution of the antibody wasapplied in an amount of 2.5 mL to single NAP-25 column, and then thefraction (3.5 mL) eluted with 3.5 mL of PBS6.5/EDTA was collected. Theresulting fraction was concentrated by the Common procedure A. Aftermeasuring the concentration of the antibody using the Common procedureB, the antibody concentration was adjusted to 20 mg/mL usingPBS6.5/EDTA.

Common Procedure D-1: Purification of Antibody-Drug Conjugate

NAP-25 column was equilibrated with any buffer selected fromcommercially available phosphate buffer (PBS7.4, Cat. No. 10010-023,Invitrogen), sodium phosphate buffer (10 mM, pH 6.0; it is referred toas PBS6.0) containing sodium chloride (137 mM), and acetate buffercontaining sorbitol (5%) (10 mM, pH 5.5; it is referred to as ABS in thespecification). Aqueous solution of the antibody-drug conjugate reactionwas applied in an amount of about 1.5 mL to the NAP-25 column, and theneluted with the buffer in an amount defined by the manufacturer tocollect the antibody fraction. The collected fraction was again appliedto the NAP-25 column and, by repeating 2 to 3 times in total the gelfiltration purification process for eluting with buffer, theantibody-drug conjugate excluding non-conjugated drug linker and alow-molecular-weight compound (tris(2-carboxyethyl)phosphinehydrochloride (TCEP), N-acetyl-L-cysteine (NAC), and dimethyl sulfoxide)was obtained. Common procedure E: Measurement of antibody concentrationin antibody-drug conjugate and average number of conjugated drugmolecules per antibody molecule.

The conjugated drug concentration in the antibody-drug conjugate can becalculated by measuring UV absorbance of an aqueous solution of theantibody-drug conjugate at two wavelengths of 280 nm and 370 nm,followed by performing the calculation shown below.

Because the total absorbance at any wavelength is equal to the sum ofthe absorbance of every light-absorbing chemical species that arepresent in a system [additivity of absorbance], when the molarabsorption coefficients of the antibody and the drug remain the samebefore and after conjugation between the antibody and the drug, theantibody concentration and the drug concentration in the antibody-drugconjugate are expressed with the following equations.

A₂₈₀=A_(D,280)+A_(A,280)=ε_(D,280)C_(D)+ε_(A,280)C_(A)  Equation (1)

A₃₇₀=A_(D,370)+A_(A,370)=ε_(D,370)C_(D)+ε_(A,370)C_(A)  Equation (2)

In the above, A₂₈₀ represents the absorbance of an aqueous solution ofthe antibody-drug conjugate at 280 nm, A₃₇₀ represents the absorbance ofan aqueous solution of the antibody-drug conjugate at 370 nm, A_(A,280)represents the absorbance of an antibody at 280 nm, A_(A,370) representsthe absorbance of an antibody at 370 nm, A_(D,280) represents theabsorbance of a conjugate precursor at 280 nm, A_(D,370) represents theabsorbance of a conjugate precursor at 370 nm, ε_(A,280) represents themolar absorption coefficient of an antibody at 280 nm, ε_(A,370)represents the molar absorption coefficient of an antibody at 370 nm,ε_(D,280) represents the molar absorption coefficient of a conjugateprecursor at 280 nm, ε_(D,370) represents the molar absorptioncoefficient of a conjugate precursor at 370 nm, C_(A) represents theantibody concentration in an antibody-drug conjugate, and C_(D)represent the drug concentration in an antibody-drug conjugate.

As for ε_(A,280), ε_(A,370), ε_(D,280), and ε_(D,370) in the above,previously prepared values (estimated value based on calculation ormeasurement value obtained by UV measurement of the compound) are used.For example, ε_(A,280) can be estimated from the amino acid sequence ofan antibody using a known calculation method (Protein Science, 1995,vol. 4, 2411-2423). ε_(A,370) is generally zero. ε_(D,280) and ε_(D,370)can be obtained based on Lambert-Beer's law (Absorbance=molarconcentration×molar absorption coefficient×cell path length) bymeasuring the absorbance of a solution in which the conjugate precursorto be used is dissolved at a certain molar concentration. By measuringA₂₈₀ and A₃₇₀ of an aqueous solution of the antibody-drug conjugate andsolving the simultaneous equations (1) and (2) using the values, C_(A)and C_(D) can be obtained. Further, by diving C_(D) by C_(A), theaverage number of conjugated drug per antibody can be obtained.

The compound represented by the formula (2) in Production method 1 isany compound represented by the following formula:

In the formula, n¹, n², n³, L², L^(P), L^(a), L^(b), and L^(c) are asalready defined, and L^(c) is a connecting position for the drug.

In an intermediate useful in producing such a compound of the presentinvention, preferably, n² is an integer of 2 to 5, L² is—NH—(CH₂CH₂O)n⁵-CH₂CH₂—C(═O)— or a single bond, n⁵ is an integer of 2 to4, L^(P) is GGFG, and —NH—(CH₂)n¹-L^(a)-L^(b)-L^(c)- is a partialstructure of —NH—CH₂CH₂—C(═O)—, NH—CH₂CH₂CH₂—C(═O)—,—NH—CH₂—O—CH₂—C(═O)—, or —NH—CH₂CH₂—O—CH₂—C(═O)—. Halogen is preferablybromine or iodine. Specific examples of these compounds can include thefollowings [herein, (maleimid-N-yl) represents a maleimidyl group(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl group)].

-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂OH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   X—CH₂—C(═O)—NH—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)

In the formula, X represents a bromine atom or an iodine atom. All ofthese bromine and iodine compounds can be preferably used as productionintermediates.

In order to secure the amount of the conjugate, a plurality ofconjugates obtained under similar production conditions to have anequivalent number of drugs (e.g., about ±1) can be mixed to prepare newlots. In this case, the average number of drugs falls between theaverage numbers of drugs in the conjugates before the mixing.

2. Production Method 2

The antibody-drug conjugate represented by the formula (1) in which theantibody is connected via an amide group to a linker and having athioether bond within the linker, specifically, a structure in which-L¹-L²- is —C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)-S—(CH₂)n⁶-C(═O)—,can be also produced by the following method.

In the formula, AB-L^(1′) represents a group which the antibody andlinker L¹ are connected and, further, the terminal of L¹ is converted toa N-maleimidyl group. This group specifically has a structure in which—(N-ly-3-diminiccuS)- in AB—C(═O)-cyc.Hex(1,4)-CH₂—(N-ly-3-diminiccuS)-is converted to a maleimidyl group. L²′ represents a HS—(CH₂)n⁶-C(═O)—group in which the terminal is a mercapto group, and AB represents theantibody.

Specifically, the antibody-drug conjugate (1) can be produced byreacting the compound (2a), which is obtainable by the method describedbelow, with the antibody (3b) which is connected to the linker having amaleimidyl group.

The antibody (3b) having a maleimidyl group can be also obtained by amethod well known in the art (Hermanson, G. T, Bioconjugate Techniques,pp. 56-136, pp. 456-493, Academic Press (1996)). Examples include: abifunctional linker, such assuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),which is capable of bonding to an amino group or a hydroxyl group andhas a maleimidyl group is allowed to react on the amino group of theligand to introduce a maleimidyl group, but it is not limited thereto.

For example, a compound having an amino group-reactive moiety and athiol group-reactive moiety bound via a linker can be preferably used.Here, the amino group-reactive moiety can be active ester, imide ester,or the like, and the thiol-reactive moiety can be maleimidyl, acetylhalide, alkyl halide, dithiopyridyl, or the like.

As a method for constructing the linker with amino group or hydroxygroup of an amino acid constituting the antibody, particularly via anamide bond with the amino group, the compound to be first reacted withthe antibody can be a compound represented by the following formula:

Q¹-L^(1a)-Q².

[In the formula, Q¹ represents (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—,(3-Sulfo-pyrrolidine-2,5-dione-N-yl)-O—C(═O)—, R^(Q)—O—C(═N)—, orO═C═N—,

L^(1a)- represents -cyc.Hex(1,4)-CH₂—, an alkylene group having 1 to 10carbon atoms, a phenylene group, (CH₂)n⁴-C(═O)—, —(CH₂)n^(4a)-NH—C(═O)—(CH₂) n^(4b)-, or (CH₂) n^(4a)-NH—C(═O)-cyc.Hex(1,4)-CH₂—,Q² represents (maleimid-N-yl), a halogen atom, or —S—S-(2-Pyridyl),R^(Q) represents an alkyl group having 1 to 6 carbon atoms, n⁴represents an integer of 1 to 8, n^(4a) represents an integer of 0 to 6,and n^(4b) represents an integer of 1 to 6.]

In the above, R^(Q) is an alkyl group having 1 to 6 carbon atoms, andmore preferably a methyl group or an ethyl group.

The alkylene group of L^(1a) may be those having 1 to carbon atoms. Thephenylene group may be any of ortho, meta, and para configurations andis more preferably a para- or meta-phenylene group.

Preferred examples of L^(1a) can include cyc.Hex (1,4)-CH₂—,—(CH₂)₅—NH—C(═O)-cyc.Hex (1,4)-CH₂—, (CH₂)₂—NH—C(═O)—CH₂—,—(CH₂)₅—NH—C(═O)—(CH₂)₂—, —CH₂—, —(CH₂)₂—, —(CH₂)₃—, —(CH₂)₅—,—(CH₂)₁₀—, -(para-Ph)-, -(meta-Ph)-, -(para-Ph)-CH(—CH₃)—,—(CH₂)₃-(meta-Ph)-, and -(meta-Ph)-NH—C(═O)—CH₂—.

Q¹ is preferably (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—, Q² is preferably(maleimid-N-yl), or —S—S-(2-Pyridyl) can be used when a disulfide bondis to be formed.

In the above, (Pyrrolidine-2,5-dione-N-yl)- is a group represented bythe following formula:

wherein the nitrogen atom as a connecting position, and(3-Sulfo-pyrrolidine-2,5-dione-N-yl)- is a group represented by thefollowing formula:

wherein the nitrogen atom is a connecting position, and this sulfonicacid is capable of forming a lithium salt, sodium salt, or potassiumsalt, and preferably sodium salt,cyc.Hex(1,4) represents a 1,4-cyclohexylene group, (maleimid-N-yl) is agroup represented by the following formula:

wherein the nitrogen atom is a connecting position, (2-Pyridyl)represents a 2-pyridyl group, (para-Ph) represents a para-phenylenegroup, and (meta-Ph) represents a meta-phenylene group.

Examples of such a compound includesulfosuccinimidyl-4-(N-maleimidylmethyl)cyclohexane-1-carboxylate(sulfo-SMCC),N-succinimidyl-4-(N-maleimidylmethyl)-cyclohexane-1-carboxy-(6-amidocaproate)(LC-SMCC), κ-maleimidyl undecanoic acid N-succinimidyl ester (KMUA),γ-maleimidyl butyric acid N-succinimidyl ester (GMBS), ε-maleimidylcaproic acid N-hydroxysuccinimide ester (EMCS),m-maleimidylbenzoyl-N-hydroxysuccinimide ester (MBS),N-(α-maleimidylacetoxy)-succinimide ester (AMAS),succinimidyl-6-(β-maleimidylpropionamide)hexanoate (SMPH),N-succinimidyl 4-(p-maleimidylphenyl)-butyrate (SMPB),N-(p-maleimidylphenyl)isocyanate (PMPI),N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyliodoacetate (SIA), N-succinimidyl bromoacetate (SBA), N-succinimidyl3-(bromoacetamide)propionate (SBAP),N-succinimidyl-3-(2-pyridodithio)propionate (SPDP), andsuccinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT).

Specifically, for example, by reacting 2 to 6 equivalents of SMCC withthe antibody (3) in a phosphate buffer of pH 6 to 7 at room temperaturefor 1 to 6 hours, the active ester of SMCC can react with the antibodyto yield the antibody (3b) having a maleimidyl group. The obtainedantibody (3b) can be purified by Common procedure D-2 described below,and used for the next reaction with the compound (2a).

Common Procedure D-2: Purification of Succinimidyl4-(N-maleimidylmethyl)-cyclohexane-1-carboxylate (SMCC)-derivatizedAntibody

NAP-25 column was equilibrated with PBS6.5/EDTA. Reaction solutioncontaining the succinimidyl4-(N-maleimidylmethyl)-cyclohexane-1-carboxylate (herein, referred to asSMCC)-derivatized antibody was applied in an amount of about 0.5 mL tothe NAP-25 column, and then eluted with the buffer in an amount definedby the manufacturer to collect the antibody fraction for purification.

The amino group of the antibody for connecting to the linker can be aN-terminal amino group and/or an amino group carried by a lysineresidue, but it is not limited thereto. Alternatively, the antibody maybe connected to the linker with ester bond formation by use of a hydroxygroup carried by a serine residue.

The reaction of the compound (2a) with the antibody (3b) connected tothe linker having a maleimidyl group can be performed in the same manneras the method for reacting the compound (2) with the antibody (3a)having a sulfhydryl group as mentioned in Production method 1.

For the antibody-drug conjugate (1) prepared, concentration, bufferexchange, purification, and identification of the antibody-drugconjugate (1) by the measurement of antibody concentration and anaverage number of conjugated drug molecules per antibody molecule can beperformed in the same manner as Production method 1.

The compound represented by the formula (3b) in Production method 2 hasthe following structure (see the following formula; in the structurethereof, “antibody —NH—” originates from an antibody).

A compound which is an intermediate for producing the antibody-drugconjugate of the present invention and has the above structure is asdescribed below (in the formula, n is an integer of 1 to 10, preferably2 to 8, and more preferably 3 to 8).

Further, examples of the compound of the present invention in which theterminal is a mercapto group can include the followings.

-   HS—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)

3. Production Method 3

The antibody-drug conjugate represented by the formula (1) in which theantibody is conjugated to the drug linker moiety via an amide bond canbe produced by a method described below. For example, as for—C(═O)—(CH₂)n⁴-C(═O)— of L¹, its active ester L^(1′), for example,(Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—(CH₂)n⁴-C(═O)—, can be preferablyused. When L² is a single bond, the antibody-drug conjugate (1) can beproduced by the following method, for example.

Specifically, the antibody-drug conjugate (1) can be produced byreacting the compound (2b), which is obtainable by the method describedbelow, with the antibody (3).

The compound (2b) is capable of connecting to the amino group orhydroxyl group of the antibody. The amino group and hydroxyl group ofthe antibody refer to, as described in Production method 2, for example,a N-terminal amino group carried by the antibody and/or an amino groupcarried by a lysine residue and a hydroxy group carried by a serineresidue, respectively, but they are not limited thereto.

The compound (2b) is activie ester composed of a N-hydroxysuccinimidylester group. Alternatively, other active esters, for example, asulfosuccinimidyl ester group, N-hydroxyphthalimidyl ester,N-hydroxysulfophthalimidyl ester, ortho-nitrophenyl ester,para-nitrophenyl ester, 2,4-dinitrophenyl ester,3-sulfonyl-4-nitrophenyl ester, 3-carboxy-4-nitrophenyl ester, andpentafluorophenyl ester, may be used.

By using 2 to 20 molar equivalents of the compound (2b) per the antibody(3) in the reaction of the compound (2b) with the antibody(3), theantibody-drug conjugate (1) in which 1 to 10 drug molecules areconjugated per antibody can be produced. Specifically, the solutioncontaining the compound (2b) dissolved therein can be added to a buffersolution containing the antibody (3) for the reaction to yield theantibody-drug conjugate (1). Herein, examples of the buffer solutionwhich may be used include sodium acetate solution, sodium phosphate, andsodium borate. pH for the reaction can be 5 to 9, and more preferablythe reaction is performed near pH 7. Examples of the solvent fordissolving the compound (2b) include an organic solvent such as dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethyl acetamide (DMA), andN-methyl-2-pyridone (NMP). It is sufficient that the organic solventsolution containing the compound (2b) dissolved therein is added at 1 to20% v/v to a buffer solution containing the antibody (3) for thereaction. The reaction temperature is 0 to 37° C., more preferably 10 to25° C., and the reaction time is 0.5 to 20 hours.

For the produced antibody-drug conjugate (1), concentration, bufferexchange, purification, and identification of the antibody-drugconjugate (1) by the measurement of antibody concentration and anaverage number of conjugated drug molecules per antibody molecule can beperformed in the same manner as Production method 1.

The moiety (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—(CH₂)n⁴-C(═O)— inProduction method 3 has the following structure.

Examples of the compound of the present invention having the abovepartial structure can include the followings.

-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)

4. Production Method 4

The compound represented by the formula (2) or (2b) as an intermediateused in the previous production method and a pharmacologicallyacceptable salt thereof can be produced by the following method, forexample.

In the formula, L^(c) is —C(═O)— and is connected to —(NH-DX) withformation of amide bond, L^(1′) represents L¹ structure in which theterminal is converted to a maleimidyl group or a haloacetyl group, or to(Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—(CH₂)n⁴-C(═O)—, and P¹, P², and P³each represents a protecting group.

The compound (6) can be produced by derivatizing the carboxylic acid (5)into an active ester, mixed acid anhydride, acid halide, or the like andreacting it with NH₂-DX [indicating exatecan; chemical name:(1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10,13(9H,15H)-dione](4) or a pharmacologically acceptable salt thereof.

Reaction reagents and conditions that are commonly used for peptidesynthesis can be employed for the reaction. There are various kinds ofactive ester. For example, it can be produced by reacting phenols suchas p-nitrophenol, N-hydroxy benzotriazole, N-hydroxy succinimide, or thelike, with the carboxylic acid (5) using a condensing agent such asN,N′-dicyclohexylcarbodiimide Or1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Further,the active ester can be also produced by a reaction of the carboxylicacid (5) with pentafluorophenyl trifluoroacetate or the like; a reactionof the carboxylic acid (5) with 1-benzotriazolyloxytripyrrolidinophosphonium hexafluorophosphite; a reaction of thecarboxylic acid (5) with diethyl cyanophosphonate (salting-in method); areaction of the carboxylic acid (5) with triphenylphosphine and2,2′-dipyridyl disulfide (Mukaiyama's method); a reaction of thecarboxylic acid (5) with a triazine derivative such as4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM); or the like. Further, the reaction can be also performed by,e.g., an acid halide method by which the carboxylic acid (5) is treatedwith acid halide such as thionyl chloride and oxalyl chloride in thepresence of a base. By reacting the active ester, mixed acid anhydride,or acid halide of the carboxylic acid (5) obtained accordingly with thecompound (4) in the presence of a suitable base in an inert solvent at−78° C. to 150° C., the compound (6) can be produced. (Meanwhile, “inertsolvent” indicates a solvent which does not inhibit a reaction for whichthe solvent is used.)

Specific examples of the base used for each step described above includecarbonate of an alkali metal or an alkali earth metal, an alkali metalalkoxide, hydroxide or hydride of an alkali metal including sodiumcarbonate, potassium carbonate, sodium ethoxide, potassium butoxide,sodium hydroxide, potassium hydroxide, sodium hydride, and potassiumhydride, organometallic base represented by an alkyl lithium includingn-butyl lithium, dialkylamino lithium including lithiumdiisopropylamide; organometallic base of bissilylamine including lithiumbis(trimethylsilyl)amide; and organic base including pyridine,2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine,N-methylmorpholine, diisopropylethylamine, anddiazabicyclo[5.4.0]undec-7-ene (DBU).

Examples of the inert solvent which is used for the reaction of thepresent invention include a halogenated hydrocarbon solvent such asdichloromethane, chloroform, and carbon tetrachloride; an ether solventsuch as tetrahydrofuran, 1,2-dimethoxyethane, and dioxane; an aromatichydrocarbon solvent such as benzene and toluene; and an amide solventsuch as N,N-dimethylformamide, N,N-dimethylacetamide, andN-methylpyrrolidin-2-one. In addition to them, a sulfoxide solvent suchas dimethyl sulfoxide and sulfolane; and a ketone solvent such asacetone and methyl ethyl ketone may be used depending on a case.

The hydroxy group, carboxy group, amino group, or the like of L^(a) andL^(b) in the compound (6) may be protected with a protecting group whichis commonly used in organic compound synthesis, as mentioned later.Specifically, examples of the protecting group for a hydroxyl groupinclude an alkoxymethyl group such as methoxymethyl group; an arylmethylgroup such as benzyl group, 4-methoxybenzyl group, and triphenylmethylgroup; an alkanoyl group such as acetyl group; an aroyl group such asbenzoyl group; and a silyl group such as tert-butyl diphenylsilyl group.Carboxy group can be protected, e.g., as an ester with an alkyl groupsuch as methyl group, ethyl group, and tert-butyl group, an allyl group,or an arylmethyl group such as benzyl group. Amino group can beprotected with a protecting group for an amino group which is generallyused for peptide synthesis, for example, an alkyloxy carbonyl group suchas tert-butyloxy carbonyl group, methoxycarbonyl group, andethoxycarbonyl group; an arylmethyl group such as allyloxycarbonyl,9-fluorenylmethyloxy carbonyl group, benzyloxy carbonyl group,paramethoxybenzyloxy carbonyl group, and para (or ortho)nitroybenzyloxycarbonyl group; an alkanoyl group such as acetyl group; an arylmethylgroup such as benzyl group and triphenyl methyl group; an aroyl groupsuch as benzoyl group; and an aryl sulfonyl group such as2,4-dinitrobenzene sulfonyl group or orthonitrobenzene sulfonyl group.Protection with and deprotection of the protecting group can beperformed according to a method commonly carried out.

As for the protecting group P¹ for the terminal amino group of thecompound (6), a protecting group for an amino group which is generallyused for peptide synthesis, for example, tert-butyloxy carbonyl group,9-fluorenylmethyloxy carbonyl group, and benzyloxy carbonyl group, canbe used. Examples of the other protecting group for an amino groupinclude an alkanoyl group such as acetyl group; an alkoxycarbonyl groupsuch as methoxycarbonyl group and ethoxycarbonyl group; an arylmethoxycarbonyl group such as paramethoxybenzyloxy carbonyl group, and para (orortho)nitroybenzyloxy carbonyl group; an arylmethyl group such as benzylgroup and triphenyl methyl group; an aroyl group such as benzoyl group;and an aryl sulfonyl group such as 2,4-dinitrobenzene sulfonyl group andorthonitrobenzene sulfonyl group. The protecting group P¹ can beselected depending on, e.g., properties of a compound having an aminogroup to be protected.

By deprotecting the protecting group P¹ for the terminal amino group ofthe compound (6) obtained, the compound (7) can be produced. Reagentsand conditions can be selected depending on the protecting group.

The compound (9) can be produced by derivatizing the peptide carboxylicacid (8) having the N terminal protected with P² into an active ester,mixed acid anhydride, or the like and reacting it with the compound (7)obtained. The reaction conditions, reagents, base, and inert solventused for forming a peptide bond between the peptide carboxylic acid (8)and the compound (7) can be suitably selected from those described forthe synthesis of the compound (6). The protecting group P² can besuitably selected from those described for the protecting group of thecompound (6), and the selection can be made based on, e.g., theproperties of the compound having an amino group to be protected. As itis generally used for peptide synthesis, by repeating sequentially thereaction and deprotection of the amino acid or peptide constituting thepeptide carboxylic acid (8) for elongation, the compound (9) can be alsoproduced.

By deprotecting P² as the protecting group for the amino group of thecompound (9) obtained, the compound (10) can be produced. Reagents andconditions can be selected depending on the protecting group.

It is possible to produce the compound (2) or (2b) by derivatizing thecarboxylic acid (11) or (11b) into an active ester, mixed acidanhydride, acid halide, or the like and reacting it with the compound(10) obtained. The reaction conditions, reagents, base, and inertsolvent used for forming a peptide bond between the carboxylic acid (11)or (11b) and the compound (10) can be suitably selected from thosedescribed for the synthesis of the compound (6).

The compound (9) can be also produced by the following method, forexample.

The compound (13) can be produced by derivatizing the peptide carboxylicacid (8) having the N terminal protected with P² into active ester,mixed acid anhydride, or the like and reacting it with the aminecompound (12) having the carboxy group protected with P³ in the presenceof a base. The reaction conditions, reagents, base, and inert solventused for forming a peptide bond between the peptide carboxylic acid (8)and the compound (12) can be suitably selected from those described forthe synthesis of the compound (6). The protecting group P² for the aminogroup of the compound (13) can be suitably selected from those describedfor the protecting group of the compound (6). As for the protectinggroup P³ for a carboxy group, a protecting group commonly used as aprotecting group for a carboxy group in organic synthetic chemistry, inparticular, peptide synthesis can be used. Specifically, it can besuitably selected from those described for the protecting group of thecompound (6), for example, esters with an alkyl group such as a methylgroup, an ethyl group, or a tert-butyl, allyl esters, and benzyl esters.In such case, it is necessary that the protecting group for an aminogroup and the protecting group for a carboxy group can be removed by adifferent method or different conditions. For example, a representativeexample includes a combination in which P² is a tert-butyloxy carbonylgroup and P³ is a benzyl group. The protecting groups can be selectedfrom the aforementioned ones depending on, e.g., the properties of acompound having an amino group and a carboxy group to be protected. Forremoval of the protecting groups, reagents and conditions can beselected depending on the protecting group.

By deprotecting the protecting group P³ for the carboxy group of thecompound (13) obtained, the compound (14) can be produced. Reagents andconditions are selected depending on the protecting group.

The compound (9) can be produced by derivatizing the compound (14)obtained into active ester, mixed acid anhydride, acid halide, or thelike and reacting with the compound (4) in the presence of a base. Forthe reaction, reaction reagents and conditions that are generally usedfor peptide synthesis can be also used, and the reaction conditions,reagents, base, and inert solvent used for the reaction can be suitablyselected from those described for the synthesis of the compound (6).

The compound (2) or (2b) can be also produced by the following method,for example.

By deprotecting the protecting group P² for the amino group of thecompound (13), the compound (15) can be produced. Reagents andconditions can be selected depending on the protecting group.

The compound (16) or (16b) can be produced by derivatizing thecarboxylic acid derivative (11) or (11b) into active ester, mixed acidanhydride, acid halide, or the like and reacting it with the compound(15) obtained in the presence of a base. The reaction conditions,reagents, base, and inert solvent used for forming an amide bond betweenthe peptide carboxylic acid (11) or (11b) and the compound (15) can besuitably selected from those described for the synthesis of the compound(6).

By deprotecting the protecting group for the carboxy group of thecompound (16) or (16b) obtained, the compound (17) or (17b) can beproduced. It can be carried out similar to deprotecting carboxy groupfor producing the compound (14).

The compound (2) or (2b) can be produced by derivatizing the compound(17) or (17b) into active ester, mixed acid anhydride, acid halide, orthe like and reacting it with the compound (4) in the presence of abase. For the reaction, reaction reagents and conditions that aregenerally used for peptide synthesis can be also used, and the reactionconditions, reagents, base, and inert solvent used for the reaction canbe suitably selected from those described for the synthesis of thecompound (6).

5. Production Method 5

The compound represented by the formula (2) of an intermediate can bealso produced by the following method.

In the formula, L^(1′) corresponds to L¹ having a structure in which theterminal is converted to a maleimidyl group or a haloacetyl group, andP⁴ represents a protecting group.

The compound (19) can be produced by derivatizing the compound (11) intoactive ester, mixed acid anhydride, or the like and reacting it with thepeptide carboxylic acid (18) having the C terminal protected with P⁴ inthe presence of a base. The reaction conditions, reagents, base, andinert solvent used for forming a peptide bond between the peptidecarboxylic acid (18) and the compound (11) can be suitably selected fromthose described for the synthesis of the compound (6). The protectinggroup P⁴ for the carboxy group of the compound (18) can be suitablyselected from those described for the protecting group of the compound(6).

By deprotecting the protecting group for the carboxy group of thecompound (19) obtained, the compound (20) can be produced. It can beperformed similar to the deprotection of the carboxy group for producingthe compound (14).

The compound (2) can be produced by derivatizing the compound (20)obtained into active ester, mixed acid anhydride, or the like andreacting it with the compound (7). For the reaction, reaction reagentsand conditions that are generally used for peptide synthesis can be alsoused, and the reaction conditions, reagents, base, and inert solventused for the reaction can be suitably selected from those described forthe synthesis of the compound (6).

6. Production Method 6

The production intermediate (2a) described in Production method 2 inwhich L²′ corresponds to L² having a structure in which the terminal isconverted to a mercaptoalkanoyl group can be produced by the followingmethod.

The compound (2a) can be produced by derivatizing the carboxylic acid(21) having a terminal mercapto group into active ester, mixed acidanhydride, or the like and reacting it with the compound (10). For thereaction, reaction reagents and conditions that are generally used forpeptide synthesis can be also used, and the reaction conditions,reagents, base, and inert solvent used for the reaction can be suitablyselected from those described for the synthesis of the compound (4).

Further, the compound (23) can be produced by derivatizing the compound(21) into active ester, mixed acid anhydride, acid halide, or the like,reacting it with the compound (15), and deprotecting the protectinggroup for the carboxy group of the compound (22) obtained.

The compound (2a) can be produced by derivatizing the compound (23) intoactive ester, mixed acid anhydride, acid halide, or the like andreacting it with the compound (4) in the presence of a base. For thereaction, reaction reagents and conditions that are generally used forpeptide synthesis can be also used, and the reaction conditions,reagents, base, and inert solvent used for the reaction can be suitablyselected from those described for the synthesis of the compound (6).

7. Production Method 7

Hereinbelow, the method for producing the compound (10c) having n¹=1,L^(a)=O, and L^(b)=CR²(—R³) in the production intermediate (10)described in Production method 4 is described in detail. The compoundrepresented by the formula (10c), a salt or a solvate thereof can beproduced according to the following method, for example.

In the formula, L^(P), R², and R³ are as defined above, L represents anacetyl group, a hydrogen atom, or the like, X and Y each represent anoligopeptide consisting of 1 to 3 amino acids, P⁵ and P⁷ each representa protecting group for an amino group, and P⁶ represents a protectinggroup for a carboxy group.

A compound represented by the formula (24) can be produced by using orapplying the method described in Japanese Patent Laid-Open No.2002-60351 or the literature (J. Org. Chem., Vol. 51, page 3196, 1986),and if necessary, by removing the protecting groups or modifying thefunctional groups. Alternatively, it can be also obtained by treating anamino acid with a protected terminal amino group or acid amide ofoligopeptide with protected amino group with aldehyde or ketone.

By reacting the compound (24) with the compound (25) having a hydroxylgroup at a temperature ranging from under cooling to room temperature inan inert solvent in the presence of an acid or a base, the compound (26)can be produced. Examples of the acid which may be used includeinorganic acid such as hydrofluoric acid, hydrochloric acid, sulfuricacid, nitric acid, phosphoric acid, and boric acid; an organic acid suchas acetic acid, citric acid, paratoluene sulfonic acid, and methanesulfonic acid; and a Lewis acid such as tetrafluoroborate, zincchloride, tin chloride, aluminum chloride, and iron chloride.Paratoluene sulfonic acid is particularly preferable. As for the base tobe used, any one of the aforementioned base can be suitably selected andused. Preferred examples thereof include an alkali metal alkoxide suchas potassium tert-butoxide, an alkali metal hydroxide such as sodiumhydroxide and potassium hydroxide; alkali metal hydride such as sodiumhydride and potassium hydride; organometallic base represented bydialkylamino lithium such as lithium diisopropylamide; andorganometallic base of bissilylamine such as lithiumbis(trimethylsilyl)amide. Examples of the solvent to be used for thereaction include an ether solvent such as tetrahydrofuran and1,4-dioxane; and an aromatic hydrocarbon solvent such as benzene andtoluene. Those solvents can be prepared as a mixture with water.Further, the protecting group for an amino group as exemplified by P⁵ isnot particularly limited if it is a group commonly used for protectionof an amino group. Representative examples include the protecting groupsfor an amino group that are described in Production method 4. However,in the present reaction, the protecting group for an amino group asexemplified by P⁵ may be cleaved off. In such case, it is necessary toperform a reaction with a suitable reagent for protecting an amino groupas it may be required.

The compound (27) can be produced by removing the protecting group P⁶ ofthe compound (26). Herein, although the representative examples of theprotecting group for a carboxy group as exemplified by P⁶ are describedin Production method 4, it is desirable in this case that the protectinggroup P⁵ for an amino group and the protecting group P⁶ for a carboxygroup are the protecting groups that can be removed by a differentmethod or different conditions. For example, a representative exampleincludes a combination in which P⁵ is a 9-fluorenylmethyloxy carbonylgroup and P⁶ is a benzyl group. The protecting groups can be selecteddepending on, e.g., the properties of a compound having an amino groupand a carboxy group to be protected. For removal of the protectinggroups, reagents and conditions are selected depending on the protectinggroup.

The compound (29) can be produced by derivatizing the carboxylic acid(27) into active ester, mixed acid anhydride, acid halide, or the likeand reacting it with the compound (4) and a pharmacologically acceptablesalt thereof to produce the compound (28) followed by removing theprotecting group P⁵ of the compound (28) obtained. For the reactionbetween the compound (4) and the carboxylic acid (27) and the reactionfor removing the protecting group P⁶, the same reagents and reactionconditions as those described for Production method 4 can be used.

The compound (10c) can be produced by reacting the compound (29) with anamino acid with protected terminal amino group or the oligopeptide (30)with protected amino group to produce the compound (9c) and removing theprotecting group P⁷ of the compound (9c) obtained. The protecting groupfor an amino group as exemplified by P⁷ is not particularly limited ifit is generally used for protection of an amino group. Representativeexamples thereof include the protecting groups for an amino group thatare described in Production method 4. For removing the protecting group,reagents and conditions are selected depending on the protecting group.For the reaction between the compound (29) and the compound (30),reaction reagents and conditions that are commonly used for peptidesynthesis can be employed. The compound (10c) produced by theaforementioned method can be derivatized into the compound (1) of thepresent invention according to the method described above.

8. Production Method 8

Hereinbelow, the method for producing the compound (2c) having n¹=1,L^(a)=O, and L^(b)=CR²(—R³) in the production intermediate (2) describedin Production method 4 is described in detail. The compound representedby the formula (2c), a salt or a solvate thereof can be producedaccording to the following method, for example.

In the formula, L¹, L², L^(P), R², and R³ are as defined above, Zrepresents an oligopeptide consisting of 1 to 3 amino acids, P⁸represents a protecting group for an amino group, and P⁹ represents aprotecting group for a carboxy group.

The compound (33) can be produced by removing the protecting group P⁸ ofthe amino acid or oligopeptide (31) with protected terminal amino groupand carboxy group to produce the compound (32) and reacting the obtainedamine form (32) with the compound (11). The protecting group for anamino group as exemplified by P⁸ is not particularly limited if it is agroup commonly used for protection of an amino group. Representativeexamples include the protecting groups for an amino group that aredescribed in Production method 4. Further, for removing the protectinggroup P⁸, reagents and conditions can be selected depending on theprotecting group. For the reaction between the compound (32) and thecarboxylic acid (11), the same reagents and reaction conditions as thosedescribed for Production method 4 can be used.

The production intermediate (2c) can be produced by removing theprotecting group P⁹ of the compound (33) to produce the compound (34)and reacting the obtained carboxylic acid (34) with the compound (29).The representative examples of the protecting group for a carboxy groupas exemplified by P⁹ are described in Production method 4. For thedeprotection reaction thereof, the same reagents and reaction conditionsas those described for Production method 4 can be used. For the reactionbetween the compound (29) and the carboxylic acid (34), reactionreagents and conditions that are generally used for peptide synthesiscan be also used. The compound (2c) produced by the aforementionedmethod can be derivatized into the compound (1) of the present inventionaccording to the method described above.

9. Production Method 9

Hereinbelow, the method for producing the compound (17c) having n¹=1,L^(a)=O, and L^(b)=CR²(—R³) in the production intermediate (17)described in Production method 4 is described in detail. The compoundrepresented by the formula (17c), a salt or a solvate thereof can bealso produced according to the following method, for example.

In the formula, L^(1′), L², L^(P), R², R³, X, Y, P⁵, P⁶, and P⁷ are asdefined above.

The compound (36) can be produced by deprotecting the protecting groupP⁵ for the amino group of the compound (26) with protected terminalamino group and carboxy group to produce the compound (35) and reactingthe obtained amine form (35) with the oligopeptide (30) with protectedterminal amino group or protected amino group. The protecting group foran amino group as exemplified by P⁵ is not particularly limited if it isa group commonly used for protection of an amino group. Representativeexamples include the protecting groups for an amino group that aredescribed in Production method 4. Further, for removing the protectinggroup P⁵, reagents and conditions can be selected depending on theprotecting group. Herein, although representative examples of theprotecting group for a carboxy group as exemplified by P⁶ and theprotecting group for an amino group as exemplified by P⁷ include theprotecting groups for a carboxy group and an amino group that aredescribed in Production method 4, it is desirable that the protectinggroup P⁶ for a carboxy group and the protecting group P⁷ for an aminogroup are the protecting groups that can be removed by the same methodor the same conditions. For example, a representative example includes acombination in which P⁶ is a benzyl ester group and P⁷ is a benzyloxycarbonyl group.

The compound (37) can be produced by removing the protecting group P⁶for the carboxy group of the compound (36) and the protecting group P⁷for the amino group of the compound (36). The compound (37) can be alsoproduced by sequentially removing the protecting group P⁶ for thecarboxy group and the protecting group P⁷ for the amino group, or thecompound (37) can be produced by removing at once both of the protectinggroups P⁶ and P⁷ that can be removed by the same method or the sameconditions.

The compound (17c) can be produced by reacting the obtained compound(37) with the compound (11). For the reaction between the compound (37)and the compound (11), the same reagents and reaction conditions asthose described for Production method 4 can be used.

In the foregoing, the compound represented by the following formula:

is described as a production intermediate useful for producing theantibody-drug conjugate of the present invention. In addition, a groupof compounds represented by the following formula: Q-(CH₂)n^(Q)-C(═O)-L^(2a)-L^(P)-NH—(CH₂) n¹-L^(a)-L^(b)-L^(c)-(NH-DX) are alsocompounds that serve as production intermediates useful for producingthe antibody-drug conjugate of the present invention.

Specifically, in the above formula, Q is (maleimid-N-yl)-, HS—,X—CH₂—C(═O)—NH—, or (pyrrolidine-2,5-dione-N-yl)-O—C(═O)—,

X is a bromine atom or an iodine atom,n^(Q) is an integer of 2 to 8,L^(2a) represents —NH—(CH₂—CH₂-0)n⁵-CH₂—CH₂—C(═O)— or a single bond,

wherein n⁵ represents an integer of 1 to 6, L^(P) represents a peptideresidue consisting of 2 to 7 amino acids,

n¹ represents an integer of 0 to 6,L^(a) represents —C(═O)—NH—, —NR¹—(CH₂)n⁷-, —O—, or a single bond,

wherein n⁷ represents an integer of 1 to 6, R¹ represents a hydrogenatom, an alkyl group having 1 to carbon atoms, —(CH₂) n⁸-COOH, or—(CH₂)n⁹-OH, n⁸ represents an integer of 1 to 4, n⁹ represents aninteger of 1 to 6,

L^(b) represents —CR²(—R³)—, —O—, —NR⁴—, or a single bond,

wherein R² and R³ each independently represent a hydrogen atom, an alkylgroup having 1 to 6 carbon atoms, —(CH₂)n^(a)-NH₂, —(CH₂)n^(b)-COOH, or—(CH₂)n^(c)-OH, R⁴ represents a hydrogen atom or an alkyl group having 1to 6 carbon atoms, n^(a) represents an integer of 0 to 6, n^(b)represents an integer of 1 to 4, n^(c) represents an integer of 1 to 4,provided that when n^(a) is 0, R² and R³ are not the same as each other,

L^(c) represents —CH₂— or —C(═O)—,(maleimid-N-yl)- is a group having a structure represented by thefollowing formula:

(in the formula, the nitrogen atom is the connecting position),(Pyrrolidine-2,5-dione-N-yl)- is a group having a structure representedby the following formula:

(in the formula, the nitrogen atom is the connecting position), —(NH-DX)is a group having a structure represented by the following formula:

(in the formula, the nitrogen atom of the amino group at position 1 isthe connecting position).

A compound in which L^(c) is —C(═O)— is preferred as a productionintermediate.

As for the peptide residue of L^(P), a compound of an amino acid residueconsisting of an amino acid selected from phenylalanine, glycine,valine, lysine, citrulline, serine, glutamic acid, and aspartic acid ispreferred as a production intermediate. Among those peptide residues, acompound in which L^(P) is a peptide residue consisting of 4 amino acidsis preferred as a production intermediate. More specifically, a compoundin which L^(P) is -GGFG- is preferred as a production intermediate.

Further, as for the —NH—(CH₂)n¹-L^(a)-L^(b)-, a compound of —NH—CH₂CH₂—,—NH—CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—,or —NH—CH₂CH₂—O—CH₂— is preferred as a production intermediate. Acompound of NH—CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—, or —NH—(CH₂)₂—O—, —CH₂—C(═O)— is more preferred.

As for n^(Q), a compound in which it is an integer of 2 to 6 ispreferred as a production intermediate.

A compound in which L^(2a) is a single bond or n⁵ is an integer of 2 to4 is preferred as a production intermediate.

When Q is (maleimid-N-yl)-, a compound in which n^(Q) is an integer of 2to 5, L^(2a) is a single bond, and —NH—(CH₂) n¹-L^(a)-L^(b)- is—NH—CH₂CH₂—, —NH—CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂CH₂—,—NH—CH₂—O—CH₂—, or NH—CH₂CH₂—O—CH₂— is preferred as a productionintermediate. A compound in which —NH—(CH₂)n¹-L^(a)-L^(b)- is—NH—CH₂CH₂—, —NH—CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—, or —NH—CH₂CH₂—O—CH₂— ismore preferred. A compound in which n^(Q) is an integer of 2 or 5 isfurther preferred.

Also, when Q is (maleimid-N-yl)-, a compound in which n^(Q) is aninteger of 2 to 5, L^(2a) is —NH—(CH₂—CH₂—O)n⁵-CH₂—CH₂—C(═O)—, n⁵ is aninteger of 2 to 4, and —NH—(CH₂)n¹-L^(a)-L^(b)- is —NH—CH₂CH₂—,—NH—CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—,or NH—CH₂CH₂—O—CH₂— is preferred as a production intermediate. Acompound in which n⁵ is an integer of or 4 is more preferred. A compoundin which —NH—(CH₂)n¹-L^(a)-L^(b)- is —NH—CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—, or—NH—CH₂CH₂—O—CH₂— is further preferred.

When Q is HS—, a compound in which n^(Q) is an integer of 2 to 5, L^(2a)is a single bond, and —NH—(CH₂) n¹-L^(a)-L^(b)- is —NH—CH₂CH₂—,—NH—CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—,or —NH—CH₂CH₂—O—CH₂— is preferred as a production intermediate. Acompound in which —NH—(CH₂)n¹-L^(a)-L^(b)- is —NH—CH₂CH₂CH₂—,—NH—CH₂—O—CH₂—, or —NH—CH₂CH₂—O—CH₂— is more preferred.

When Q is X—CH₂—C(═O)—NH—, a compound in which X is a bromine atom ispreferred as a production intermediate. A compound in which n^(Q) is aninteger of 2 to 8 is preferred, also a compound in which L^(2a) is asingle bond is preferred, and a compound in which—NH—(CH₂)n¹-L^(a)-L^(b)- is —NH—CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—, or—NH—CH₂CH₂—O—CH₂— is preferred as a production intermediate.

When Q is (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—, a compound in whichn^(Q) is an integer of 2 to 5, L^(2a) is a single bond, and—NH—(CH₂)n¹-L^(a)-L^(b)- is —NH—CH₂CH₂—, —NH—CH₂CH₂CH₂—,—NH—CH₂CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—, or—NH—CH₂CH₂—O—CH₂— is preferred as a production intermediate. A compoundin which —NH—(CH₂)n¹-L^(a)-L^(b)- is —NH—CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—, or—NH—CH₂CH₂—O—CH₂— is more preferred.

More specifically, the followings are compounds preferred as productionintermediates.

-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX)-   (maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   HS—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   Br—CH₂—C(═O)—NH—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)-   (Pyrrolidine-2,5-dione-N-yl)-O—C(═O)—CH₂CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)

Meanwhile, the antibody-drug conjugate of the present invention, when itis left in air or recrystallized, may absorb moisture to have adsorptionwater or turn into a hydrate, and such a compound and a salt containingwater are also included in the present invention.

A compound labeled with various radioactive or non-radioactive isotopesis also included in the present invention. One or more atomsconstituting the antibody-drug conjugate of the present invention maycontain an atomic isotope at non-natural ratio. Examples of the atomicisotope include deuterium (²H), tritium (³H), iodine-125 (¹²⁵I), andcarbon-14 (¹⁴C). Further, the compound of the present invention may beradioactive-labeled with a radioactive isotope such as tritium (³H),iodine-125 (¹²⁵I), carbon-14 (¹⁴C), copper-64 (⁶⁴Cu), zirconium-89(⁸⁹Zr), iodine-124 (¹²⁴I), fluorine-18 (¹⁸F), indium-111 (¹¹¹I),carbon-11 (¹¹C) and iodine-131 (¹³¹I). The compound labeled with aradioactive isotope is useful as a therapeutic or prophylactic agent, areagent for research such as an assay reagent and an agent for diagnosissuch as an in vivo diagnostic imaging agent. Without being related toradioactivity, any isotope variant type of the antibody-drug conjugateof the present invention is within the scope of the present invention.

[Drugs]

The antibody-drug conjugate of the present invention exhibits acytotoxic activity against cancer cells, and thus, it can be used as adrug, particularly as a therapeutic agent and/or prophylactic agent forcancer.

Examples of the cancer type to which the antibody-drug conjugate of thepresent invention is applied include lung cancer, kidney cancer,urothelial cancer, colorectal cancer, prostate cancer, glioblastomamultiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma,liver cancer, bladder cancer, stomach cancer, or esophageal cancer,however, it is not limited to them as long as it is a cancer cellexpressing, in a cancer cell as a treatment subject, a protein which theantibody within the antibody-drug conjugate can recognize.

The antibody-drug conjugate of the present invention can be preferablyadministered to a mammal, but it is more preferably administered to ahuman.

Substances used in a pharmaceutical composition containing antibody-drugconjugate of the present invention can be suitably selected and appliedfrom formulation additives or the like that are generally used in theart, in view of the dosage or administration concentration.

The antibody-drug conjugate of the present invention can be administeredas a pharmaceutical composition containing at least one pharmaceuticallysuitable ingredient.

For example, the pharmaceutical composition above typically contains atleast one pharmaceutical carrier (for example, sterilized liquid). forexample, water and oil (petroleum oil and oil of animal origin, plantorigin, or synthetic origin (the oil may be, for example, peanut oil,soybean oil, mineral oil, sesame oil or the like)). Water is a moretypical carrier when the pharmaceutical composition above isintravenously administered. Saline solution, an aqueous dextrosesolution, and an aqueous glycerol solution can be also used as a liquidcarrier, in particular, for an injection solution. A suitablepharmaceutical vehicle is known in the art. If desired, the compositionabove may also contain a trace amount of a moisturizing agent, anemulsifying agent, or a pH buffering agent. Examples of suitablepharmaceutical carrier are disclosed in “Remington's PharmaceuticalSciences” by E. W. Martin. The formulations correspond to anadministration mode.

Various delivery systems are known and they can be used foradministering the antibody-drug conjugate of the present invention.Examples of the administration route include intradermal, intramuscular,intraperitoneal, intravenous, and subcutaneous routes, but not limitedthereto. The administration can be made by injection or bolus injection,for example. According to a specific preferred embodiment, theadministration of the antibody-drug conjugate is performed by injection.Parenteral administration is a preferred administration route.

According to a representative embodiment, the pharmaceutical compositionis prescribed, as a pharmaceutical composition suitable for intravenousadministration to human, according to the conventional procedures. Thecomposition for intravenous administration is typically a solution in asterile and isotonic aqueous buffer solution. If necessary, the drug maycontain a solubilizing agent and local anesthetics to alleviate pain atinjection site (for example, lignocaine). Generally, the ingredientabove is provided individually as any one of lyophilized powder or ananhydrous concentrate contained in a container which is obtained bysealing in an ampoule or a sachet having an amount of the active agentor as a mixture in a unit dosage form. When the drug is to beadministered by injection, it may be administered from an injectionbottle containing water or saline of sterile pharmaceutical grade. Whenthe drug is administered by injection, an ampoule of sterile water orsaline for injection may be provided such that the aforementionedingredients are admixed with each other before administration.

The pharmaceutical composition of the present invention may be apharmaceutical composition containing only the antibody-drug conjugateof the present invention or a pharmaceutical composition containing theantibody-drug conjugate and at least one cancer treating agent otherthan the conjugate. The antibody-drug conjugate of the present inventioncan be administered with other cancer treating agent. The anti-cancereffect may be enhanced accordingly. Another anti-cancer agent used forsuch purpose may be administered to an individual simultaneously with,separately from, or subsequently to the antibody-drug conjugate, and itmay be administered while varying the administration interval for each.Examples of the cancer treating agent include abraxane, carboplatin,cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed,sorafenib, vinorelbine, drugs described in International Publication No.WO 2003/038043, LH-RH analogues (leuprorelin, goserelin, or the like),estramustine phosphate, estrogen antagonist (tamoxifen, raloxifene, orthe like), and an aromatase inhibitor (anastrozole, letrozole,exemestane, or the like), but it is not limited as long as it is a drughaving an antitumor activity.

The pharmaceutical composition can be formulated into a lyophilizationformulation or a liquid formulation as a formulation having desiredcomposition and required purity. When formulated as a lyophilizationformulation, it may be a formulation containing suitable formulationadditives that are used in the art. Also for a liquid formulation, itcan be formulated as a liquid formulation containing various formulationadditives that are used in the art.

Composition and concentration of the pharmaceutical composition may varydepending on administration method. However, the antibody-drug conjugatecontained in the pharmaceutical composition of the present invention canexhibit the pharmaceutical effect even at a small dosage when theantibody-drug conjugate has higher affinity for an antigen, that is,higher affinity (=lower Kd value) in terms of the dissociation constant(that is, Kd value) for the antigen. Thus, for determining dosage of theantibody-drug conjugate, the dosage can be determined in view of asituation relating to the affinity between the antibody-drug conjugateand antigen. When the antibody-drug conjugate of the present inventionis administered to a human, for example, about 0.001 to 100 mg/kg can beadministered once or administered several times with an interval of onetime for 1 to 180 days.

EXAMPLES

The present invention is specifically described in view of the examplesshown below. However, the present invention is not limited to them.Further, it is by no means interpreted in a limited sense. Further,unless specifically described otherwise, the reagent, solvent, andstarting material described in the specification can be easily obtainedfrom a commercial supplier.

Reference Example 1 M30-H1-L4 Antibody

Of humanized antibodies of an anti-B7-H3 antibody, an antibody composedof a heavy chain consisting of an amino acid sequence described in aminoacid positions 20 to 471 in SEQ ID NO: 9 and a light chain consisting ofan amino acid sequence described in amino acid positions 21 to 233 inSEQ ID NO: 16 was produced in accordance with a method known in the artto yield humanized anti-B7-H3 antibody designated as an M30-H1-L4antibody (or simply referred to as “M30-H1-L4”).

Reference Example 2 M30-H1-L4P Antibody

The modification of a glycan bonded to the M30-H1-L4 antibody obtainedabove was regulated by defucosylation in accordance with a method knownin the art to yield antibody with the regulated modification of a glycandesignated as an M30-H1-L4P antibody (or simply referred to as“M30-H1-L4P”).

Reference Example 3 Anti-CD30 Antibody

An anti-CD30 antibody was produced with reference to NationalPublication of International Patent Application No. 2005-506035. Itssequence is shown in SEQ ID NOs: 27 and 28.

Reference Example 4 Anti-CD33 Antibody

An anti-CD33 antibody was produced with reference to Japanese PatentLaid-Open No. 8-48637. Its sequence is shown in SEQ ID NOs: 29 and 30.

Reference Example 5 Anti-CD70 Antibody

An anti-CD70 antibody was produced with reference to NationalPublication of International Patent Application No. 2008-538292. Itssequence is shown in SEQ ID NOs: 31 and 32.

Example 14-Amino-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]butanamide

Process 1: tert-Butyl(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)carbamate

4-(tert-Butoxycarbonylamino)butanoic acid (0.237 g, 1.13 mmol) wasdissolved in dichloromethane (10 mL), N-hydroxysuccinimide (0.130 g,1.13 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (0.216 g, 1.13 mmol) were added, and stirred for 1 hour.The reaction solution was added dropwise to an N,N-dimethylformamidesolution (10 mL) charged with mesylate of the compound (4) (0.500 g,0.94 mmol) and triethylamine (0.157 mL, 1.13 mmol), and stirred at roomtemperature for 1 day. The solvent was removed under reduced pressureand the residue obtained were purified by silica gel columnchromatography [chloroform-chloroform:methanol=8:2 (v/v)] to yield thetitled compound (0.595 g, quantitative).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.2 Hz), 1.31 (9H, s), 1.58(1H, t, J=7.2 Hz), 1.66 (2H, t, J=7.2 Hz), 1.82-1.89 (2H, m), 2.12-2.21(3H, m), 2.39 (3H, s), 2.92 (2H, t, J=6.5 Hz), 3.17 (2H, s), 5.16 (1H,d, J=18.8 Hz), 5.24 (1H, d, J=18.8 Hz), 5.42 (2H, s), 5.59-5.55 (1H, m),6.53 (1H, s), 6.78 (1H, t, J=6.3 Hz), 7.30 (1H, s), 7.79 (1H, d, J=11.0Hz), 8.40 (1H, d, J=8.6 Hz).

MS (APCI) m/z: 621 (M+H)⁺

Process 2:4-Amino-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]butanamide

The compound (0.388 g, 0.61 mmol) obtained in Process 1 above wasdissolved in dichloromethane (9 mL). Trifluoroacetic acid (9 mL) wasadded and it was stirred for 4 hours. The solvent was removed underreduced pressure and the residues obtained were purified by silica gelcolumn chromatography [chloroform-partitioned organic layer ofchloroform:methanol water=7:3:1 (v/v/v)] to yield trifluoroacetate ofthe titled compound (0.343 g, quantitative). This compound was confirmedin the tumor of a cancer-bearing mouse that received the antibody-drugconjugate (13) or (14).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.2 Hz), 1.79-1.92 (4H, m),2.10-2.17 (2H, m), 2.27 (2H, t, J=7.0 Hz), 2.40 (3H, s), 2.80-2.86 (2H,m), 3.15-3.20 (2H, m), 5.15 (1H, d, J=18.8 Hz), 5.26 (1H, d, J=18.8 Hz),5.42 (2H, s), 5.54-5.61 (1H, m), 6.55 (1H, s), 7.32 (1H, s), 7.72 (3H,brs), 7.82 (1H, d, J=11.0 Hz), 8.54 (1H, d, J=8.6 Hz).

MS (APCI) m/z: 521 (M+H)⁺

Example 2 Antibody-Drug Conjugate (1)

Process 1:N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanylglycine (0.081 g, 0.19mmol) was dissolved in dichloromethane (3 mL), N-hydroxysuccinimide(0.021 g, 0.19 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (0.036 g, 0.19 mmol) were added and then stirred for 3.5hours. The reaction solution was added dropwise to anN,N-dimethylformamide solution (1.5 mL) charged with the compound (0.080g, 0.15 mmol) of Example 1, and stirred at room temperature for 4 hours.The solvent was removed under reduced pressure and the residues obtainedwere purified by silica gel column chromatography [chloroform-chloroformmethanol=: 2 (v/v)] to yield the titled compound (0.106 g, 73%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.36 (9H, s), 1.71(2H, m), 1.86 (2H, t, J=7.8 Hz), 2.15-2.19 (4H, m), 2.40 (3H, s), 2.77(1H, dd, J=12.7, 8.8 Hz), 3.02 (1H, dd, J=14.1, 4.7 Hz), 3.08-3.11 (2H,m), 3.16-3.19 (2H, m), 3.54 (2H, d, J=5.9 Hz), 3.57-3.77 (4H, m),4.46-4.48 (1H, m), 5.16 (1H, d, J=19.2 Hz), 5.25 (1H, d, J=18.8 Hz),5.42 (2H, s), 5.55-5.60 (1H, m), 6.53 (1H, s), 7.00 (1H, t, J=6.3 Hz),7.17-7.26 (5H, m), 7.31 (1H, s), 7.71 (1H, t, J=5.7 Hz), 7.80 (1H, d,J=11.0 Hz), 7.92 (1H, t, J=5.7 Hz), 8.15 (1H, d, J=8.2 Hz), 8.27 (1H, t,J=5.5 Hz), 8.46 (1H, d, J=8.2 Hz).

MS (APCI) m/z: 939 (M+H)⁺

Process 2:Glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamidetrifluoroacetate

The compound (1.97 g, 2.10 mmol) obtained in Process 1 above wasdissolved in dichloromethane (7 mL). After adding trifluoroacetic acid(7 mL), it was stirred for 1 hour. The solvent was removed under reducedpressure, and it was charged with toluene for azeotropic distillation.The residues obtained were purified by silica gel column chromatography[chloroform-partitioned organic layer of chloroform:methanol:water=7:3:1(v/v/v)] to yield the titled compound (1.97 g, 99%).

¹H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J=7.4 Hz), 1.71-1.73 (2H, m),1.82-1.90 (2H, m), 2.12-2.20 (4H, m), 2.40 (3H, s), 2.75 (1H, dd,J=13.7, 9.4 Hz), 3.03-3.09 (3H, m), 3.18-3.19 (2H, m), 3.58-3.60 (2H,m), 3.64 (1H, d, J=5.9 Hz), 3.69 (1H, d, J=5.9 Hz), 3.72 (1H, d, J=5.5Hz), 3.87 (1H, dd, J=16.8, 5.9 Hz), 4.50-4.56 (1H, m), 5.16 (1H, d,J=19.2 Hz), 5.25 (1H, d, J=18.8 Hz), 5.42 (2H, s), 5.55-5.60 (1H, m),7.17-7.27 (5H, m), 7.32 (1H, s), 7.78-7.81 (2H, m), 7.95-7.97 (3H, m),8.33-8.35 (2H, m), 8.48-8.51 (2H, m).

MS (APCI) m/z: 839 (M+H)⁺

Process 3:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

To an N,N-dimethylformamide (1.2 mL) solution of the compound (337 mg,0.353 mmol) obtained in Process 2 above, triethylamine (44.3 mL, 0.318mmol) and N-succinimidyl 6-maleimide hexanoate (119.7 mg, 0.388 mmol)were added and stirred at room temperature for 1 hour. The solvent wasremoved under reduced pressure and the residues obtained were purifiedby silica gel column chromatography [chloroform chloroform:methanol=5:1(v/v)] to yield the titled compound as a pale yellow solid (278.0 mg,76%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.3 Hz), 1.12-1.22 (2H, m),1.40-1.51 (4H, m), 1.66-1.76 (2H, m), 1.80-1.91 (2H, m), 2.05-2.21 (6H,m), 2.39 (3H, s), 2.79 (1H, dd, J=14.0, 9.8 Hz), 2.98-3.21 (5H, m),3.55-3.77 (8H, m), 4.41-4.48 (1H, m), 5.15 (1H, d, J=18.9 Hz), 5.24 (1H,d, J=18.9 Hz), 5.40 (1H, d, J=17.1 Hz), 5.44 (1H, d, J=17.1 Hz),5.54-5.60 (1H, m), 6.53 (1H, s), 6.99 (2H, s), 7.20-7.27 (5H, m), 7.30(1H, s), 7.70 (1H, t, J=5.5 Hz), 7.80 (1H, d, J=11.0 Hz), 8.03 (1H, t,J=5.8 Hz), 8.08 (1H, t, J=5.5 Hz), 8.14 (1H, d, J=7.9 Hz), 8.25 (1H, t,J=6.1 Hz), 8.46 (1H, d, J=8.5 Hz).

MS (APCI) m/z: 1032 (M+H)⁺

Process 4: Antibody-Drug Conjugate (1)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.61mLmg⁻¹cm⁻¹ was used) described in Production method 1. The solution(1.25 mL) was placed in a 1.5 mL polypropylene tube and charged with anaqueous solution of 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.)(0.025 mL; 3.0 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (Nacalai Tesque, Inc.;0.0625 mL). After confirming that the solution had pH of 7.4±0.1, thedisulfide bond at hinge part in the antibody was reduced by incubatingat 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (Sigma-Aldrich Co. LLC; 0.109 mL) and a dimethyl sulfoxidesolution containing 10 mM of the compound obtained in above Process 3(0.039 mL; 4.6 equivalents per antibody molecule) to the above solutionat room temperature, it was stirred by using a tube rotator (MTR-103,manufactured by AS ONE Corporation) for conjugating the drug linker tothe antibody at room temperature for 40 minutes. Next, an aqueoussolution (0.008 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was addedthereto and stirred at room temperature to terminate the raction of druglinker for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate. After that, the solution was concentrated bythe Common procedure A.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 13.02 mg/mL, antibody yield: 9.1 mg (73%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.4.

Example 3 Antibody-Drug Conjugate (2)

Process 1: Antibody-Drug Conjugate (2)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (4.0 mL) was collected into a 15 mLtube and charged with an aqueous solution of 10 mM TCEP (Tokyo ChemicalIndustry Co., Ltd.) (0.118 mL; 4.6 equivalents per antibody molecule)and an aqueous solution of 1 M dipotassium hydrogen phosphate (NacalaiTesque, Inc.; 0.200 mL). After confirming that the solution had pH of7.4±0.1, the disulfide bond at hinge part in the antibody was reduced byincubatingat 37° C. for 1 hour. Conjugation between antibody and druglinker: After incubating the above solution for 10 minutes at 22° C., adimethyl sulfoxide solution (0.236 mL; 9.2 equivalents per antibodymolecule) containing 10 mM of the compound obtained in Process 3 ofExample 2 was added thereto and incubated for conjugating the druglinker to the antibody at 22° C. for 40 minutes. Next, an aqueoussolution (0.00471 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was addedthereto and incubated to terminate the raction of drug linker at 22° C.for another 20 minutes. Purification: The above solution was subjectedto purification using the Common procedure D-1 (ABS was used as buffersolution) described in Production method 1 to yield 17.5 mL of asolution containing the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,εA,280=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 1.80 mg/mL, antibody yield: 26.1 mg (65%), andaverage number of conjugated drug molecules (n) per antibody molecule:5.9.

Example 4 Antibody-Drug Conjugate (3)

Process 1: Antibody-Drug Conjugate (3)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.61mLmg⁻¹cm⁻¹ was used) described in Production method 1. The solution(1.25 mL) was placed in a 1.5 mL polypropylene tube and charged with anaqueous solution of 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.)(0.051 mL; 6.0 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (Nacalai Tesque, Inc.;0.0625 mL). After confirming that the solution had pH of 7.4±0.1, thedisulfide bond at hinge part in the antibody was reduced by incubatingat 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (Sigma-Aldrich Co. LLC; 0.067 mL) and a dimethyl sulfoxidesolution containing 10 mM of the compound obtained in Process 3 ofExample 2 (0.085 mL; 10.0 equivalents per antibody molecule) to theabove solution at room temperature, it was stirred by using a tuberotator (MTR-103, manufactured by AS ONE Corporation) for conjugatingthe drug linker to the antibody at room temperature for 60 minutes.Next, an aqueous solution (0.013 mL) of 100 mM NAC (Sigma-Aldrich Co.LLC) was added thereto and stirred to terminate the raction of druglinker at room temperature for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 1.67 mg/mL, antibody yield: 10.02 mg (80%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.3.

Example 5 Antibody-Drug Conjugate (4)

Process 1: Antibody-Drug Conjugate (4)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.61mLmg⁻¹cm⁻¹ was used) described in Production method 1. The solution(1.25 mL) was placed in a 1.5 mL polypropylene tube and charged with anaqueous solution of 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.)(0.051 mL; 6.0 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (Nacalai Tesque, Inc.;0.0625 mL). After confirming that the solution had pH of 7.4±0.1, thedisulfide bond at hinge part in the antibody was reduced by incubatingat 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (Sigma-Aldrich Co. LLC; 0.025 mL) and a dimethyl sulfoxidesolution containing 10 mM of the compound obtained in Process 3 ofExample 2 (0.127 mL; 15.0 equivalents per antibody molecule) to theabove solution at room temperature, it was stirred by using a tuberotator (MTR-103, manufactured by AS ONE Corporation) for conjugatingthe drug linker to the antibody at room temperature for 60 minutes.Next, an aqueous solution (0.019 mL) of 100 mM NAC (Sigma-Aldrich Co.LLC) was added thereto and stirred to terminate the raction of druglinker at room temperature for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate. After that, the solution was concentrated bythe Common procedure A.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 1.19 mg/mL, antibody yield: 7.14 mg (57%), andaverage number of conjugated drug molecules (n) per antibody molecule:7.5.

Example 6 Antibody-Drug Conjugate (5)

Almost the whole amounts of the antibody-drug conjugates of Examples 4and 5 were mixed and the solution was concentrated by the Commonprocedure A to yield the titled antibody-drug conjugate.

Antibody concentration: 10.0 mg/mL, antibody yield: 15.37 mg, andaverage number of conjugated drug molecules (n) per antibody molecule:6.7.

Example 7 Antibody-Drug Conjugate (6)

Process 1: Antibody-Drug Conjugate (6)

Reduction of the antibody: The anti-CD30 antibody produced in ReferenceExample 3 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.75 was used) and Common procedure C-1 described inProduction method 1. The solution (1.0 mL) was collected into a 2 mLtube and charged with an aqueous solution of 10 mM TCEP (Tokyo ChemicalIndustry Co., Ltd.) (0.0297 mL; 4.6 equivalents per antibody molecule)and an aqueous solution of 1 M dipotassium hydrogen phosphate (NacalaiTesque, Inc.; 0.050 mL). After confirming that the solution had pH of7.4±0.1, the disulfide bond at hinge part in the antibody was reduced byincubatingat 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating the abovesolution at 22° C. for 10 minutes, a dimethyl sulfoxide solution (0.0593mL; 9.2 equivalents per antibody molecule) containing 10 mM of thecompound obtained in Process 3 of Example 2 was added thereto andincubated for conjugating the drug linker to the antibody at 22° C. for40 minutes. Next, an aqueous solution (0.0119 mL; 18.4 equivalents perantibody molecule) of 100 mM NAC (Sigma-Aldrich Co. LLC) was addedthereto and incubated to terminate the raction of drug linker at 22° C.for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=270400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 0.99 mg/mL, antibody yield: 5.94 mg (59%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.3.

Example 8 Antibody-Drug Conjugate (7)

Process 1: Antibody-Drug Conjugate (7)

Reduction of the antibody: The anti-CD30 antibody produced in ReferenceExample 3 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.75 was used) and Common procedure C-1 described inProduction method 1. The solution (1.0 mL) was collected into a 2 mLtube and charged with an aqueous solution of 30 mM TCEP (Tokyo ChemicalIndustry Co., Ltd.) (0.0148 mL; 6.9 equivalents per antibody molecule)and an aqueous solution of 1 M dipotassium hydrogen phosphate (NacalaiTesque, Inc.; 0.050 mL). After confirming that the solution had pH of7.4±0.1, the disulfide bond at hinge part in the antibody was reduced byincubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating the abovesolution for 10 minutes at 22° C., a dimethyl sulfoxide solution (0.0297mL; 13.8 equivalents per antibody molecule) containing 30 mM of thecompound obtained in Process 3 of Example 2 was added thereto andincubated for 40 minutes at 22° C. for conjugating the drug linker tothe antibody. Next, an aqueous solution (0.0178 mL; 27.6 equivalents perantibody molecule) of 100 mM NAC (Sigma-Aldrich Co. LLC) was addedthereto and incubated to terminate the raction of drug linker at 22° C.for another 20 minutes. Purification: The above solution was subjectedto purification using the Common procedure D-1 (ABS was used as buffersolution) described in Production method to yield 6 mL of a solutioncontaining the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=270400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 0.99 mg/mL, antibody yield: 5.94 mg (59%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.8.

Example 9 Antibody-Drug Conjugate (8)

Process 1: Antibody-Drug Conjugate (8)

Reduction of the antibody: The anti-CD33 antibody produced in ReferenceExample 4 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.66 was used) and Common procedure C-1 described inProduction method 1. The solution (1.0 mL) was collected into a 2 mLtube and charged with an aqueous solution of 10 mM TCEP (Tokyo ChemicalIndustry Co., Ltd.) (0.0297 mL; 4.6 equivalents per antibody molecule)and an aqueous solution of 1 M dipotassium hydrogen phosphate (NacalaiTesque, Inc.; 0.050 mL). After confirming that the solution had pH of7.4±0.1, the disulfide bond at hinge part in the antibody was reduced byincubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating the abovesolution for 10 minutes at 22° C., a dimethyl sulfoxide solution (0.0593mL; 9.2 equivalents per antibody molecule) containing 10 mM of thecompound obtained in Process 3 of Example 2 was added thereto andincubated for conjugating the drug linker to the antibody at 22° C. for40 minutes. Next, an aqueous solution (0.0119 mL; 18.4 equivalents perantibody molecule) of 100 mM NAC (Sigma-Aldrich Co. LLC) was addedthereto and incubated to terminate the raction of drug linker at 22° C.for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=256400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 1.06 mg/mL, antibody yield: 6.36 mg (64%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.4.

Example 10 Antibody-Drug Conjugate (9)

Process 1: Antibody-Drug Conjugate (9)

Reduction of the antibody: The anti-CD33 antibody produced in ReferenceExample 4 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.66 was used) and Common procedure C-1 described inProduction method 1. The solution (1.0 mL) was collected into a 2 mLtube and charged with an aqueous solution of 30 mM TCEP (Tokyo ChemicalIndustry Co., Ltd.) (0.0148 mL; 6.9 equivalents per antibody molecule)and an aqueous solution of 1 M dipotassium hydrogen phosphate (NacalaiTesque, Inc.; 0.050 mL). After confirming that the solution had pH of7.4±0.1, the disulfide bond at hinge part in the antibody was reduced byincubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating the abovesolution for 10 minutes at 22° C., a dimethyl sulfoxide solution (0.0297mL; 13.8 equivalents per antibody molecule) containing 30 mM of thecompound obtained in Process 3 of Example 2 was added thereto andincubated for conjugating the drug linker to the antibody at 22° C. for40 minutes. Next, an aqueous solution (0.0178 mL; 27.6 equivalents perantibody molecule) of 100 mM NAC (Sigma-Aldrich Co. LLC) was addedthereto and incubated to terminate the raction of drug linker at 22° C.for another 20 minutes. Purification: The above solution was subjectedto purification using the Common procedure D-1 (ABS was used as buffersolution) described in Production method to yield 6 mL of a solutioncontaining the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=256400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 0.95 mg/mL, antibody yield: 5.70 mg (57%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.7.

Example 11 Antibody-Drug Conjugate (10)

Process 1: Antibody-Drug Conjugate (10)

Reduction of the antibody: The anti-CD70 antibody produced in ReferenceExample 5 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.69 was used) and Common procedure C-1 described inProduction method 1. The solution (1.0 mL) was collected into a 2 mLtube and charged with an aqueous solution of 10 mM TCEP (Tokyo ChemicalIndustry Co., Ltd.) (0.0297 mL; 4.6 equivalents per antibody molecule)and an aqueous solution of 1 M dipotassium hydrogen phosphate (NacalaiTesque, Inc.; 0.050 mL). After confirming that the solution had pH of7.4±0.1, the disulfide bond at hinge part in the antibody was reduced byincubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating the abovesolution at 22° C. for 10 minutes, a dimethyl sulfoxide solution (0.0593mL; 9.2 equivalents per antibody molecule) containing 10 mM of thecompound obtained in Process 3 of Example 2 was added thereto andincubated for conjugating the drug linker to the antibody at 22° C. for40 minutes. Next, an aqueous solution (0.0119 mL; 18.4 equivalents perantibody molecule) of 100 mM NAC (Sigma-Aldrich Co. LLC) was addedthereto and incubated to terminate the raction of drug linker at 22° C.for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=262400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 1.00 mg/mL, antibody yield: 6.00 mg (60%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.2.

Example 12 Antibody-Drug Conjugate (11)

Process 1: Antibody-Drug Conjugate (11)

Reduction of the antibody: The anti-CD70 antibody produced in ReferenceExample 5 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.69 was used) and Common procedure C-1 described inProduction method 1. The solution (1.0 mL) was collected into a 2 mLtube and charged with an aqueous solution of 30 mM TCEP (Tokyo ChemicalIndustry Co., Ltd.) (0.0148 mL; 6.9 equivalents per antibody molecule)and an aqueous solution of 1 M dipotassium hydrogen phosphate (NacalaiTesque, Inc.; 0.050 mL). After confirming that the solution had pH of7.4±0.1, the disulfide bond at hinge part in the antibody was reduced byincubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating the abovesolution at 22° C. for 10 minutes, a dimethyl sulfoxide solution (0.0297mL; 13.8 equivalents per antibody molecule) containing 30 mM of thecompound obtained in Process 3 of Example 2 was added thereto andincubated for 40 minutes at 22° C. for conjugating the drug linker tothe antibody. Next, an aqueous solution (0.0178 mL; 27.6 equivalents perantibody molecule) of 100 mM NAC (Sigma-Aldrich Co. LLC) was addedthereto and incubated to terminate the raction of drug linker at 22° C.for another 20 minutes. Purification: The above solution was subjectedto purification using the Common procedure D-1 (ABS was used as buffersolution) described in Production method to yield 6 mL of a solutioncontaining the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=262400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 0.96 mg/mL, antibody yield: 5.76 mg (58%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.6.

Example 13 Antibody-Drug Conjugate (12)

Process 1:N-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The compound (80 mg, 0.084 mmol) obtained in Process 2 of Example 2 wasreacted in the same manner as Process 3 of Example 2 by usingN-succinimidyl 3-maleimide propioate (24.6 mg, 0.0924 mmol) instead ofN-succinimidyl 6-maleimide hexanoate to yield the titled compound as apale yellow solid (60.0 mg, 73%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.89 (3H, t, J=7.3 Hz), 1.70-1.78 (2H, m),1.81-1.94 (2H, m), 2.12-2.23 (4H, m), 2.42 (3H, s), 2.81 (1H, dd,J=13.7, 9.8 Hz), 3.01-3.15 (3H, m), 3.16-3.23 (2H, m), 3.30-3.35 (1H,m), 3.58-3.71 (6H, m), 3.71-3.79 (1H, m), 4.44-4.51 (1H, m), 5.19 (1H,d, J=19.0 Hz), 5.27 (1H, d, J=19.0 Hz), 5.43 (1H, d, J=17.6 Hz), 5.47(1H, d, J=17.6 Hz), 5.57-5.63 (1H, m), 6.56 (1H, s), 7.02 (2H, s),7.17-7.22 (1H, m), 7.22-7.30 (5H, m), 7.34 (1H, s), 7.73 (1H, t, J=5.6Hz), 7.83 (1H, d, J=10.7 Hz), 8.08 (1H, t, J=5.6 Hz), 8.15 (1H, d, J=7.8Hz), 8.30 (2H, dt, J=18.7, 5.7 Hz), 8.49 (1H, d, J=8.8 Hz).

MS (APCI) m/z: 990 (M+H)⁺

Process 2: Antibody-Drug Conjugate (12)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 1 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 4 of Example 2. Antibodyconcentration: 12.16 mg/mL, antibody yield: 8.5 mg (68%), and averagenumber of conjugated drug molecules (n) per antibody molecule: 3.4.

Example 14 Antibody-Drug Conjugate (13)

Process 1:N-{3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]propanoyl}glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The compound (100 mg, 0.119 mmol) obtained in Process 2 of Example 2 wasreacted in the same manner as Process 3 of Example 2 by usingdiisopropylethylamine (20.8 μL, 0.119 mmol) instead of triethylamine andN-succinimidyl3-(2-(2-(3-maleinimidepropanamide)ethoxy)ethoxy)propanoate (50.7 mg,0.119 mmol) instead of N-succinimidyl 6-maleimide hexanoate to yield thetitled compound as a pale yellow solid (66.5 mg, 48%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.85 (3H, t, J=7.4 Hz), 1.65-1.74 (2H, m),1.77-1.90 (2H, m), 2.07-2.19 (4H, m), 2.30 (2H, t, J=7.2 Hz), 2.33-2.36(2H, m), 2.38 (3H, s), 2.76 (1H, dd, J=13.7, 9.8 Hz), 2.96-3.18 (9H, m),3.42-3.44 (4H, m), 3.53-3.76 (10H, m), 4.43 (1H, td, J=8.6, 4.7 Hz),5.14 (1H, d, J=18.8 Hz), 5.23 (1H, d, J=18.8 Hz), 5.38 (1H, d, J=17.2Hz), 5.42 (1H, d, J=17.2 Hz), 5.52-5.58 (1H, m), 6.52 (1H, s), 6.98 (2H,s), 7.12-7.17 (1H, m), 7.18-7.25 (4H, m), 7.29 (1H, s), 7.69 (1H, t,J=5.5 Hz), 7.78 (1H, d, J=11.3 Hz), 7.98-8.03 (2H, m), 8.11 (1H, d,J=7.8 Hz), 8.16 (1H, t, J=5.7 Hz), 8.23 (1H, t, J=5.9 Hz), 8.44 (1H, d,J=9.0 Hz). MS (APCI) m/z: 1149 (M+H)⁺

Process 2: Antibody-Drug Conjugate (13)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 1 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 4 of Example 2. Antibodyconcentration: 12.76 mg/mL, antibody yield: 8.9 mg (71%), and averagenumber of conjugated drug molecules (n) per antibody molecule: 3.4.

Example 15 Antibody-Drug Conjugate (14)

Process 1: Antibody-Drug Conjugate (14)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 1 of Example 14, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 4.

Antibody concentration: 1.60 mg/mL, antibody yield: 9.60 mg (77%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.1.

Example 16 Antibody-Drug Conjugate (15)

Process 1: Antibody-Drug Conjugate (15)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 1 of Example 14, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 5.

Antibody concentration: 1.64 mg/mL, antibody yield: 9.84 mg (79%), andaverage number of conjugated drug molecules (n) per antibody molecule:7.1.

Example 17 Antibody-Drug Conjugate (16)

Almost the whole amounts of the antibody-drug conjugates of Examples 15and 16 were mixed and the solution was concentrated by the Commonprocedure A to yield the titled antibody-drug conjugate.

Antibody concentration: 10.0 mg/mL, antibody yield: 17.30 mg, andaverage number of conjugated drug molecules (n) per antibody molecule:6.5.

Example 18 Antibody-Drug Conjugate (17)

Process 1: Antibody-Drug Conjugate (17)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (100 mL, 1 g of the antibody) wasplaced in a 250 mL flask and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (2.43 mL; 3.6 equivalents perantibody molecule) and further with an aqueous solution of 1 Mdipotassium hydrogen phosphate (5 mL). After confirming that thesolution had pH near 7.4 by using a pH meter, the disulfide bond athinge part in the antibody was reduced by incubating at 37° C. for 1hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (2.14 mL) and a dimethyl sulfoxide solution containing 10 mMof the compound obtained in Process 1 of Example 14 (3.51 mL; 5.2equivalents per antibody molecule) to the above solution at roomtemperature, it was stirred with a stirrer for conjugating the druglinker to the antibody in a water bath at 15° C. for 130 minutes. Next,an aqueous solution (0.547 mL) of 100 mM NAC was added thereto andfurther incubated to terminate the raction of drug linker at roomtemperature for 20 minutes.

Purification: The above solution was subjected to ultrafiltrationpurification using an ultrafiltration apparatus composed of anultrafiltration membrane (Merck Japan, Pellicon XL Cassette, Biomax 50KDa), a tube pump (Cole-Parmer International, MasterFlex Pump model77521-40, Pump Head model 7518-00), and a tube (Cole-ParmerInternational, MasterFlex Tube L/S16). Specifically, while ABS was addeddropwise (a total of 800 mL) as a buffer solution for purification tothe reaction solution, ultrafiltration purification was performed forremoving unconjugated drug linkers and other low-molecular-weightreagents, also replacing the buffer solution with ABS, and furtherconcentrating the solution, to yield about 70 mL of a solutioncontaining the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=4964 (measured value), and ε_(D,370)=18982(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 14.5 mg/mL, antibody yield: 1.0 g (about 100%),and average number of conjugated drug molecules (n) per antibodymolecule: 3.5.

Example 19 Antibody-Drug Conjugate (18)

Process 1: Antibody-Drug Conjugate (18)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (5 mL, 50 mg of the antibody) wasplaced in a 15 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.135 mL; 4 equivalents perantibody molecule). After confirming that the solution had pH near 7.4by using a pH meter, the disulfide bond at hinge part in the antibodywas reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (0.064 mL) and a dimethyl sulfoxide solution containing 10 mMof the compound obtained in Process 1 of Example 14 (0.219 mL; 6.5equivalents per antibody molecule) to the above solution, it wasincubated for conjugating the drug linker to the antibody in a waterbath at 15° C. for 90 minutes. Next, an aqueous solution (0.033 mL; 9.8equivalents per antibody molecule) of 100 mM NAC was added thereto andincubated to terminate the raction of drug linker at room temperaturefor another 20 minutes. Purification: The above solution was subjectedto purification using the Common procedure D-1 (ABS was used as buffersolution) described in Production method to yield 19 mL of a solutioncontaining the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=4964 (measured value), and ε_(D,370)=18982(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 2.17 mg/mL, antibody yield: 41 mg (82%), andaverage number of conjugated drug molecules (n) per antibody molecule:5.0.

Example 20 Antibody-Drug Conjugate (19)

Process 1: Antibody-Drug Conjugate (19)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (4 mL, 40 mg of the antibody) wasplaced in a 15 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.140 mL; 5.2 equivalents perantibody molecule). After confirming that the solution had pH near 7.4by using a pH meter, the disulfide bond at hinge part in the antibodywas reduced by incubating at 37° C. for 1 hour. Conjugation betweenantibody and drug linker: After adding a dimethyl sulfoxide solutioncontaining 10 mM of the compound obtained in Process 1 of Example 14(0.232 mL; 8.6 equivalents per antibody molecule) to the above solution,it was incubated for conjugating the drug linker to the antibody in awater bath at 15° C. for 90 minutes. Next, an aqueous solution (0.035mL; 12.9 equivalents per antibody molecule) of 100 mM NAC was addedthereto and incubated to terminate the raction of drug linker at roomtemperature for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 13 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=4964 (measured value), and ε_(D,370)=18982(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 2.36 mg/mL, antibody yield: 31 mg (77%), andaverage number of conjugated drug molecules (n) per antibody molecule:5.9.

Example 21 Antibody-Drug Conjugate (20)

Process 1: Antibody-Drug Conjugate (20)

Reduction of the antibody: The M30-H1-L4 antibody produced in ReferenceExample 1 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (1.25 mL, 12.5 mg of the antibody)was placed in a 1.5 mL tube and charged with an aqueous solution of 10mM TCEP (Tokyo Chemical Industry Co., Ltd.) (0.0287 mL; 3.4 equivalentsper antibody molecule) and an aqueous solution of 1 M dipotassiumhydrogen phosphate (Nacalai Tesque, Inc.; 0.0625 mL). After confirmingthat the solution had pH of 7.4±0.1, the disulfide bond at hinge part inthe antibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (0.0267 mL) and a dimethyl sulfoxide solution containing 10 mMof the compound obtained in Process 1 of Example 14 (0.0439 mL; 5.2equivalents per antibody molecule) to the above solution at roomtemperature, it was incubated for conjugating the drug linker to theantibody in a water bath at 15° C. for 1 hour. Next, an aqueous solution(0.0066 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was added thereto andincubated to terminate the raction of drug linker at room temperaturefor another 20 minutes. Purification: The above solution was subjectedto purification using the Common procedure D-1 (ABS was used as buffersolution) described in Production method to yield 6 mL of a solutioncontaining the titled antibody-drug conjugate. After that, the solutionwas concentrated by the Common procedure A.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=4964 (measured value), and ε_(D,370)=18982(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 10.0 mg/mL, antibody yield: 8.7 mg (70%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.5.

Example 22 Antibody-Drug Conjugate (21)

Process 1: Antibody-Drug Conjugate (21)

Reduction of the antibody: The M30-H1-L4 antibody produced in ReferenceExample 1 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (1.25 mL, 12.5 mg of the antibody)was placed in a 1.5 mL tube and charged with an aqueous solution of 10mM TCEP (Tokyo Chemical Industry Co., Ltd.) (0.0439 mL; 5.2 equivalentsper antibody molecule) (0.0287 mL; 3.4 equivalents per antibodymolecule) and an aqueous solution of 1 M dipotassium hydrogen phosphate(Nacalai Tesque, Inc.; 0.0625 mL). After confirming that the solutionhad pH of 7.4±0.1, the disulfide bond at hinge part in the antibody wasreduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process1 of Example 14 (0.0726 mL; 8.6 equivalents per antibody molecule) tothe above solution at room temperature, it was incubated for conjugatingthe drug linker to the antibody in a water bath at 15° C. for 1 hour.Next, an aqueous solution (0.011 mL) of 100 mM NAC (Sigma-Aldrich Co.LLC) was added thereto and incubated to terminate the raction of druglinker at room temperature for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate. After that, the solution was concentrated bythe Common procedure A.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=4964 (measured value), and ε_(D,370)=18982(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 10.0 mg/mL, antibody yield: 8.3 mg (66%), andaverage number of conjugated drug molecules (n) per antibody molecule:5.5.

Example 23 Antibody-Drug Conjugate (22)

Process 1: Antibody-Drug Conjugate (22)

Reduction of the antibody: The anti-CD30 antibody produced in ReferenceExample 3 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.75 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (0.4 mL, 4 mg of the antibody) wasplaced in a 1.5 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.0065 mL; 2.5 equivalents perantibody molecule). The disulfide bond at hinge part in the antibody wasreduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (0.0098 mL) and a dimethyl sulfoxide solution containing 10 mMof the compound obtained in Process 1 of Example 14 (0.0116 mL; 4.5equivalents per antibody molecule) to the above solution at roomtemperature, it was stirred by using a tube rotator (MTR-103,manufactured by AS ONE Corporation) for conjugating the drug linker tothe antibody at room temperature for 1 hour. Next, an aqueous solution(0.0017 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was added thereto andfurther incubated to terminate the raction of drug linker at roomtemperature for 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method 1 to yield 2.5 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=270400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=4964 (measured value), and ε_(D,370)=18982(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 0.86 mg/mL, antibody yield: 2.2 mg (54%), andaverage number of conjugated drug molecules (n) per antibody molecule:2.5.

Example 24 Antibody-Drug Conjugate (23)

Process 1: Antibody-Drug Conjugate (23)

Reduction of the antibody: The anti-CD30 antibody produced in ReferenceExample 3 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.75 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (0.35 mL, 3.5 mg of the antibody)was placed in a 1.5 mL tube and charged with an aqueous solution of 10mM TCEP (Tokyo Chemical Industry Co., Ltd.) (0.0113 mL; 5 equivalentsper antibody molecule). The disulfide bond at hinge part in the antibodywas reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process1 of Example 14 (0.0204 mL; 9 equivalents per antibody molecule) andpropylene glycol (Kanto Chemical Co., Inc., 0.18 mL) to the abovesolution at room temperature, it was stirred by using a tube rotator(MTR-103, manufactured by AS ONE Corporation) for conjugating the druglinker to the antibody at room temperature for 1 hour. Next, an aqueoussolution (0.0031 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was addedthereto and further incubated to terminate the raction of drug linker atroom temperature for 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method 1 to yield 2.5 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=270400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=4964 (measured value), and ε_(D,370)=18982(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 0.41 mg/mL, antibody yield: 1.0 mg (29%), andaverage number of conjugated drug molecules (n) per antibody molecule:7.1.

Example 25 Antibody-Drug Conjugate (24)

Process 1: Antibody-Drug Conjugate (24)

Reduction of the antibody: The anti-CD33 antibody produced in ReferenceExample 4 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.66 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (0.4 mL, 4 mg of the antibody) wasplaced in a 1.5 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.0065 mL; 2.5 equivalents perantibody molecule) and an aqueous solution of 1 M dipotassium hydrogenphosphate (Nacalai Tesque, Inc.; 0.0058 mL). After confirming that thesolution had pH of 7.0±0.1, the disulfide bond at hinge part in theantibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (0.0101 mL) and a dimethyl sulfoxide solution containing 10 mMof the compound obtained in Process 1 of Example 14 (0.0116 mL; 4.5equivalents per antibody molecule) to the above solution at roomtemperature, it was stirred by using a tube rotator (MTR-103,manufactured by AS ONE Corporation) for conjugating the drug linker tothe antibody at room temperature for 1 hour. Next, an aqueous solution(0.0017 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was added thereto andfurther incubated to terminate the raction of drug linker at roomtemperature for 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method 1 to yield 2.5 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=256400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=4964 (measured value), and ε_(D,370)=18982(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 1.25 mg/mL, antibody yield: 3.1 mg (78%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.7.

Example 26 Antibody-Drug Conjugate (25)

Process 1: Antibody-Drug Conjugate (25)

Reduction of the antibody: The anti-CD33 antibody produced in ReferenceExample 4 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.66 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (0.4 mL, 4 mg of the antibody) wasplaced in a 1.5 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.0129 mL; equivalents perantibody molecule) and an aqueous solution of 1 M dipotassium hydrogenphosphate (Nacalai Tesque, Inc.; 0.006 mL). After confirming that thesolution had pH of 7.0±0.1, the disulfide bond at hinge part in theantibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process1 of Example 14 (0.0233 mL; 9 equivalents per antibody molecule) to theabove solution at room temperature, it was stirred by using a tuberotator (MTR-103, manufactured by AS ONE Corporation) for conjugatingthe drug linker to the antibody at room temperature for 1 hour. Next, anaqueous solution (0.0035 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) wasadded thereto and further incubated to terminate the raction of druglinker at room temperature for 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method 1 to yield 2.5 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=256400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=4964 (measured value), and ε_(D,370)=18982(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 1.17 mg/mL, antibody yield: 2.9 mg (73%), andaverage number of conjugated drug molecules (n) per antibody molecule:7.3.

Example 27 Antibody-Drug Conjugate (26)

Process 1: Antibody-Drug Conjugate (26)

Reduction of the antibody: The anti-CD70 antibody produced in ReferenceExample 5 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.69 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (0.4 mL, 4 mg of the antibody) wasplaced in a 1.5 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.0065 mL; 2.5 equivalents perantibody molecule) and an aqueous solution of 1 M dipotassium hydrogenphosphate (Nacalai Tesque, Inc.; 0.0058 mL). After confirming that thesolution had pH of 7.0±0.1, the disulfide bond at hinge part in theantibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (0.0101 mL) and a dimethyl sulfoxide solution containing 10 mMof the compound obtained in Process 1 of Example 14 (0.0116 mL; 4.5equivalents per antibody molecule) to the above solution at roomtemperature, it was stirred by using a tube rotator (MTR-103,manufactured by AS ONE Corporation) for conjugating the drug linker tothe antibody at room temperature for 1 hour. Next, an aqueous solution(0.0017 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was added thereto andfurther incubated to terminate the raction of drug linker at roomtemperature for 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method 1 to yield 2.5 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=262400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=4964 (measured value), and ε_(D,370)=18982(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 1.14 mg/mL, antibody yield: 2.9 mg (71%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.8.

Example 28 Antibody-Drug Conjugate (27)

Process 1: Antibody-Drug Conjugate (27)

Reduction of the antibody: The anti-CD70 antibody produced in ReferenceExample 5 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.69 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (0.4 mL, 4 mg of the antibody) wasplaced in a 1.5 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.0129 mL; equivalents perantibody molecule) and an aqueous solution of 1 M dipotassium hydrogenphosphate (Nacalai Tesque, Inc.; 0.006 mL). After confirming that thesolution had pH of 7.0±0.1, the disulfide bond at hinge part in theantibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution (0.0233 mL; 9 equivalents per antibody molecule)containing 10 mM of the compound obtained in Process 1 of Example 14 tothe above solution at room temperature, it was stirred by using a tuberotator (MTR-103, manufactured by AS ONE Corporation) for conjugatingthe drug linker to the antibody at room temperature for 1 hour. Next, anaqueous solution (0.0035 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) wasadded thereto and further incubated to terminate the raction of druglinker at room temperature for 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method 1 to yield 2.5 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,28)=262400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=4964 (measured value), and ε_(D,370)=18982(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 1.13 mg/mL, antibody yield: 2.8 mg (71%), andaverage number of conjugated drug molecules (n) per antibody molecule:7.4.

Example 29 Antibody-Drug Conjugate (28)

Process 1:N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxo-16-azanonadecan-1-oyl]glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The compound (90 mg, 0.107 mmol) obtained in Process 2 of Example 2 wasreacted in the same manner as Process 3 of Example 2 by usingdiisopropylethylamine (18.7 μL, 0.107 mmol) instead of triethylamine andN-succinimidyl1-maleinimide-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oate (55.1 mg,0.107 mmol) instead of N-succinimidyl 6-maleimide hexanoate to yield thetitled compound as a pale yellow solid (50 mg, 37%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.85 (3H, t, J=7.2 Hz), 1.64-1.74 (2H, m),1.77-1.90 (2H, m), 2.06-2.19 (4H, m), 2.27-2.32 (2H, m), 2.33-2.37 (2H,m), 2.38 (3H, s), 2.72-2.80 (3H, m), 2.96-3.19 (6H, m), 3.39-3.48 (10H,m), 3.52-3.75 (10H, m), 4.39-4.48 (1H, m), 5.14 (1H, d, J=18.8 Hz), 5.23(1H, d, J=18.8 Hz), 5.38 (1H, d, J=17.0 Hz), 5.42 (1H, d, J=17.0 Hz),5.52-5.58 (1H, m), 6.52 (1H, s), 6.98 (1H, s), 7.13-7.24 (5H, m), 7.29(1H, s), 7.69 (1H, t, J=5.5 Hz), 7.78 (1H, d, J=10.9 Hz), 7.98-8.03 (2H,m), 8.10 (1H, d, J=7.8 Hz), 8.16 (1H, t, J=5.7 Hz), 8.23 (1H, t, J=5.7Hz), 8.44 (1H, d, J=8.6 Hz).

MS (APCI) m/z: 1237 (M+H)⁺

Process 2: Antibody-Drug Conjugate (28)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.61mLmg⁻¹cm⁻¹ was used) described in Production method 1. The solution(1.25 mL) was placed in a 1.5 mL polypropylene tube and charged with anaqueous solution of 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.)(0.025 mL; 3.0 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (Nacalai Tesque, Inc.;0.0625 mL). After confirming that the solution had pH of 7.4±0.1, thedisulfide bond at hinge part in the antibody was reduced by incubatingat 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (Sigma-Aldrich Co. LLC; 0.102 mL) and a dimethyl sulfoxidesolution containing 10 mM of the compound obtained in above Process 1(0.047 mL; 5.5 equivalents per antibody molecule) to the above solutionat room temperature, it was stirred by using a tube rotator (MTR-103,manufactured by AS ONE Corporation) for conjugating the drug linker tothe antibody at room temperature for 40 minutes. Next, an aqueoussolution (0.009 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was addedthereto and stirred to terminate the raction of drug linker at roomtemperature for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate. After that, the solution was concentrated bythe Common procedure A.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 13.60 mg/mL, antibody yield: 9.5 mg (76%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.3.

Example 30 Antibody-Drug Conjugate (29)

Process 1:N-(tert-butoxycarbonyl)-β-alanylglycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The compound (0.839 g, 1.00 mmol) obtained in Process 2 of Example 2 wasreacted in the same manner as Process 1 of Example 1 by usingN-(tert-butoxycarbonyl)-β-alanine instead of4-(tert-butoxycarbonylamino)butanoic acid. The crude product obtainedwas used in the next process without purification.

Process 2:β-Alanylglycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The crude product obtained in Process 1 above was reacted in the samemanner as Process 2 of Example 2 to yield the titled compound as a paleyellow solid (0.610 g, 67%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.67-1.77 (2H, m),1.79-1.92 (2H, m), 2.09-2.22 (4H, m), 2.40 (3H, s), 2.46-2.55 (2H, m),2.82-2.73 (1H, m), 2.95-3.13 (5H, m), 3.14-3.21 (2H, m), 3.55-3.80 (6H,m), 4.44-4.52 (1H, m), 5.20 (2H, dd, J=35.0, 19.0 Hz), 5.42 (2H, s),5.53-5.60 (1H, m), 6.54 (1H, s), 7.14-7.28 (5H, m), 7.31 (1H, s), 7.67(2H, brs), 7.72-7.78 (1H, m), 7.80 (1H, d, J=11.0 Hz), 8.10-8.17 (2H,m), 8.29 (1H, t, J=5.9 Hz), 8.42 (1H, t, J=5.7 Hz), 8.47 (1H, d, J=8.6Hz).

Process 3:N-(bromoacetyl)-β-alanylglycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

To a dichloromethane (4.5 mL) solution of 2-bromoacetic acid (96.3 mg,0.693 mmol), N-hydroxysuccinimide (79.7 mg, 0.693 mmol) and1,3-diisopropylcarbodiimide (0.107 mL, 0.693 mmol) were added andstirred at room temperature. The reaction solution was added to anN,N-dimethylformamide (4.5 mL) solution of the compound (473 mg, 0.462mmol) obtained in Process 2 above and triethylamine (0.154 mL, 1.11mmol) at 0° C. and stirred at room temperature for 1 hour. The reactionsolution was purified by silica gel column chromatography [elutionsolvent:chloroform chloroform:methanol=85:15 (v/v)]. The obtained solidwas washed with chloroform:methanol:diethyl ether mixed solvent to yieldthe titled compound as a pale yellow solid (191 mg, 40%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.67-1.77 (2H, m),1.79-1.92 (2H, m), 2.08-2.22 (4H, m), 2.33 (2H, t, J=7.0 Hz), 2.40 (3H,s), 2.74-2.83 (1H, m), 2.99-3.12 (3H, m), 3.14-3.21 (2H, m), 3.24-3.30(2H, m), 3.56-3.77 (6H, m), 3.82 (2H, s), 4.41-4.51 (1H, m), 5.20 (2H,q, J=18.9 Hz), 5.42 (2H, s), 5.54-5.60 (1H, m), 6.54 (1H, s), 7.15-7.27(5H, m), 7.31 (1H, s), 7.69-7.74 (1H, m), 7.80 (1H, d, J=10.9 Hz), 8.06(1H, t, J=5.7 Hz), 8.13 (1H, d, J=7.8 Hz), 8.21-8.34 (3H, m), 8.46 (1H,d, J=8.6 Hz).

MS (ESI) m/z: 1030, 1032 (M+H)⁺

Process 4: Antibody-Drug Conjugate (29)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.61mLmg⁻¹cm⁻¹ was used) described in Production method 1. The solution(1.25 mL) was placed in a 1.5 mL polypropylene tube and charged with anaqueous solution of 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.)(0.025 mL; 3.0 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (Nacalai Tesque, Inc.;0.0625 mL). After confirming that the solution had pH of 7.4±0.1, thedisulfide bond at hinge part in the antibody was reduced by incubatingat 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (Sigma-Aldrich Co. LLC; 0.09 mL) and a dimethyl sulfoxidesolution containing 10 mM of the compound obtained in Process 3 (0.059mL; 7.0 equivalents per antibody molecule) to the above solution at roomtemperature, it was stirred by using a tube rotator (MTR-103,manufactured by AS ONE Corporation) for conjugating the drug linker tothe antibody at room temperature for 40 minutes. Next, an aqueoussolution (0.009 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was addedthereto and stirred to terminate the raction of drug linker at roomtemperature for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate. After that, the solution was concentrated bythe Common procedure A.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 13.9 mg/mL, antibody yield: 9.7 mg (78%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.2.

Example 31 Antibody-Drug Conjugate (30)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 3 of Example 30, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 4.

Antibody concentration: 1.94 mg/mL, antibody yield: 11.64 mg (93%), andaverage number of conjugated drug molecules (n) per antibody molecule:5.6.

Example 32 Antibody-Drug Conjugate (31)

Process 1: Antibody-Drug Conjugate (31)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 3 of Example 30, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 5.

Antibody concentration: 1.90 mg/mL, antibody yield: 11.40 mg (91%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.7.

Example 33 Antibody-Drug Conjugate (32)

Almost the whole amounts of the antibody-drug conjugates of Examples 31and 32 were mixed and the solution was concentrated by the Commonprocedure A to yield the titled antibody-drug conjugate.

Antibody concentration: 10.0 mg/mL, antibody yield: 21.06 mg, andaverage number of conjugated drug molecules (n) per antibody molecule:6.0.

Example 34 Antibody-Drug Conjugate (33)

Process 1: tert-Butyl4-({N⁶-(tert-butoxycarbonyl)-N²-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysyl}amino)butanoate

To an N,N-dimethylformamide (10.0 mL) solution ofN^(ε)-(tert-butoxycarbonyl)-N^(α)-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine(1.00 g, 2.14 mmol), N-hydroxysuccinimide (0.370 g, 3.20 mmol), andtert-butyl 4-aminobutanoic acid ester hydrochloride (0.830 g, 4.27mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(0.610 g, 3.20 mmol) and N,N-diisopropylethylamine (0.410 ml, 2.35 mmol)were added and stirred at room temperature for 3 days. The reactionsolution was diluted with ethyl acetate and washed with an aqueoussolution of 10% citric acid and a saturated aqueous solution of sodiumhydrogen carbonate, and saturated brine, and then the organic layer wasdried over anhydrous magnesium sulfate. The solvent was removed underreduced pressure to yield the titled compound as a colorless solid (1.35quantitative).

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.14-1.42 (4H, m), 1.36 (9H, s), 1.37 (9H,s), 1.48-1.67 (4H, m), 2.18 (2H, t, J=7.6 Hz), 2.84-2.93 (2H, m),2.99-3.11 (2H, m), 3.84-3.94 (1H, m), 4.18-4.30 (3H, m), 6.76 (1H, t,J=5.4 Hz), 7.33 (2H, t, J=7.3 Hz), 7.39-7.45 (3H, m), 7.73 (2H, dd,J=7.3, 2.7 Hz), 7.85-7.92 (3H, m).

Process 2: Tert-butyl4-{[N⁶-(tert-butoxycarbonyl)-L-lysyl]amino}butanoate

To an N,N-dimethylformamide (8.00 mL) solution of the compound (1.35 g,2.22 mmol) obtained in Process 1 above, piperidine (2.00 mL) was addedand stirred at room temperature for 1.5 hours. The solvent was removedunder reduced pressure to yield a mixture containing the titledcompound. The mixture was used for the next reaction without furtherpurification.

Process 3:N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N⁶-(tert-butoxycarbonyl)-N-(4-tert-butoxy-4-oxobutyl)-L-lysinamide

To an N,N-dimethylformamide (30.0 mL) solution of the mixture (2.22mmol) obtained in Process 2 above,N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valine (1.13 g, 3.32 mmol),N-hydroxysuccinimide (0.310 g, 2.66 mmol), and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.550 g,2.88 mmol) were added and stirred at room temperature for 18 hours. Thereaction solution was diluted with ethyl acetate and washed with asaturated aqueous solution of sodium hydrogen carbonate and saturatedbrine, and then the organic layer was dried over anhydrous magnesiumsulfate. The solvent was removed under reduced pressure and the residuesobtained were purified by silica gel column chromatography[chloroform-chloroform:methanol=1 (v/v)] to yield the titled compound asa colorless solid (0.363 g, 23%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.84 (6H, t, J=6.0 Hz), 1.12-1.64 (8H, m),1.34 (9H, s), 1.38 (9H, s), 1.90-2.04 (1H, m), 2.17 (2H, t, J=7.3 Hz),2.79-2.90 (2H, m), 2.99-3.09 (2H, m), 3.83-3.91 (1H, m), 4.08-4.44 (4H,m), 6.71 (1H, t, J=5.4 Hz), 7.32 (2H, t, J=7.3 Hz), 7.42 (3H, t, J=7.3Hz), 7.74 (2H, t, J=7.0 Hz), 7.85-7.91 (4H, m).

MS (ESI) m/z: 709 (M+H)⁺

Process 4:N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N-(3-carboxypropyl)-L-lysinamideFormate

To the compound (0.363 mg, 0.512 mmol) obtained in Process 3 above,formic acid (10.0 ml) was added and stirred at room temperature for 4hours. The solvent was removed under reduced pressure to yield thetitled compound. The compound was used for the next reaction withoutfurther purification.

Process 5:N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N⁶-(tert-butoxycarbonyl)-N-(3-carboxypropyl)-L-lysinamide

To 1,4-dioxane (5.00 mL) suspension of the compound (0.512 mmol)obtained in Process 4 above, a saturated aqueous solution of sodiumhydrogen carbonate (20.0 ml) and di-tert-butyl dicarbonate (0.178 ml,0.769 mmol) were added and stirred at room temperature for 3 hours. Thereaction solution was diluted with ethyl acetate and washed with anaqueous solution of 10% citric acid and saturated brine, and then theorganic layer was dried over anhydrous magnesium sulfate. The solventwas removed under reduced pressure to yield the titled compound as acolorless solid (0.295 g, 88%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.84 (6H, t, J=6.7 Hz), 1.13-1.39 (4H, m),1.35 (9H, s), 1.48-1.62 (4H, m), 1.91-2.04 (1H, m), 2.20 (2H, t, J=7.3Hz), 2.80-2.89 (2H, m), 2.99-3.11 (2H, m), 3.87 (1H, dd, J=8.5, 6.7 Hz),4.06-4.35 (4H, m), 6.71 (1H, t, J=6.0 Hz), 7.32 (2H, t, J=7.6 Hz),7.39-7.46 (3H, m), 7.74 (2H, t, J=7.6 Hz), 7.83-7.94 (4H, m).

MS (ESI) m/z: 653 (M+H)⁺

Process 6:N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N⁶-(tert-butoxycarbonyl)-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)-L-lysinamide

Mesylate of the compound (4) (0.240 g, 0.452 mmol) was reacted in thesame manner as Process 1 of Example 1 by using the compound (0.295 g,0.452 mmol) obtained in Process 5 above instead of4-(tert-butoxycarbonylamino)butanoic acid to yield the titled compoundas a pale orange solid (0.208 g, 43%). MS (ESI) m/z: 1071 (M+H)⁺

Process 7:L-Valyl-N⁶-(tert-butoxycarbonyl)-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)-L-lysinamide

The compound (0.208 g, 0.194 mmol) obtained in Process 6 above wasreacted in the same manner as Process 2 to yield a mixture containingthe titled compound. The mixture was used for the next reaction withoutfurther purification.

Process 8:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁶-(tert-butoxycarbonyl)-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)-L-lysinamide

The mixture (0.194 mmol) obtained in Process 7 above was reacted in thesame manner as Process 3 of Example 2 to yield the titled compound as apale yellow solid (0.133 g, 56%).

¹H-NMR (400 MHz, DMSO-d₅) δ: 0.77 (6H, t, J=5.7 Hz), 0.87 (3H, t, J=7.3Hz), 1.14-1.71 (10H, m), 1.35 (9H, s), 1.77-1.95 (3H, m), 2.02-2.23 (7H,m), 2.40 (3H, s), 2.84 (3H, q, J=6.4 Hz), 3.05 (2H, d, J=6.7 Hz), 3.17(2H, s), 3.26-3.39 (3H, m), 4.01-4.16 (2H, m), 5.15 (1H, d, J=18.7 Hz),5.24 (1H, d, J=18.7 Hz), 5.36-5.48 (2H, m), 5.51-5.60 (1H, m), 6.52 (1H,s), 6.72 (1H, t, J=6.0 Hz), 6.99 (2H, s), 7.31 (1H, s), 7.71-7.85 (5H,m), 8.41 (1H, d, J=9.1 Hz).

MS (ESI) m/z: 1041 (M+H)⁺

Process 9:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-1,-valyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)-L-lysinamidetrifluoroacetate

To a dichloromethane (10.0 ml) solution of the compound (0.110 mg, 0.106mmol) obtained in Process 8 above, trifluoroacetic acid (4.00 ml) wasadded and stirred at room temperature for 5 hours. The solvent wasremoved under reduced pressure to yield the titled compound as a paleyellow solid (70.0 mg, 64%).

¹H-NMR (400 MHz, DMSO-d₅) δ: 0.76-0.81 (6H, m), 0.87 (3H, t, J=7.3 Hz),1.12-1.31 (4H, m), 1.39-1.56 (8H, m), 1.57-1.74 (3H, m), 1.79-1.96 (3H,m), 2.06-2.18 (7H, m), 2.40 (3H, s), 2.70-2.80 (2H, m), 3.01-3.10 (2H,m), 3.13-3.22 (2H, m), 4.04 (1H, t, J=7.6 Hz), 4.10-4.20 (1H, m), 5.15(1H, d, J=18.7 Hz), 5.24 (1H, d, J=18.7 Hz), 5.36-5.47 (2H, m),5.52-5.60 (1H, m), 6.53 (1H, s), 7.00 (2H, s), 7.32 (1H, s), 7.61 (3H,brs), 7.75-7.88 (4H, m), 8.43 (1H, d, J=8.5 Hz).

MS (ESI) m/z: 941 (M+H)⁺

Process 10: Antibody-Drug Conjugate (33)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 9 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 2 of Example 29. Antibodyconcentration: 12.0 mg/mL, antibody yield: 8.4 mg (67%), and averagenumber of conjugated drug molecules (n) per antibody molecule: 3.2.

Example 35 Antibody-Drug Conjugate (34)

Process 1:N-(3-sulfanylpropanoyl)glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The compound (84.0 mg, 0.100 mmol) obtained in Process 2 of Example 2was reacted in the same manner as Process 3 of Example 2 by usingN-succinimidyl 3-mercaptopropionate instead of N-succinimidyl6-maleimide hexanoate to yield the titled compound as a pale yellowsolid (61.2 mg, 66%).

¹H-NMR (DMSO-D₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.77-1.66 (2H, m), 1.79-1.92(2H, m), 2.07-2.24 (4H, m), 2.31-2.47 (3H, m), 2.40 (3H, s), 2.59-2.69(2H, m), 2.78 (1H, dd, J=13.7, 9.8 Hz), 2.98-3.13 (3H, m), 3.14-3.23(2H, m), 3.54-3.79 (6H, m), 4.40-4.50 (1H, m), 5.20 (2H, dd, J=36.8,19.2 Hz), 5.36-5.47 (2H, m), 5.52-5.63 (1H, m), 6.54 (1H, s), 7.14-7.28(5H, m), 7.31 (1H, s), 7.68-7.74 (1H, m), 7.80 (1H, d, J=10.9 Hz),8.03-8.09 (1H, m), 8.13 (1H, d, J=7.8 Hz), 8.19-8.29 (2H, m), 8.46 (1H,d, J=8.6 Hz).

MS (ESI) m/z: 927 (M+H)⁺

Process 2: Antibody-Drug Conjugate (34)

SMCC derivatization of antibody: The M30-H1-L4P antibody produced inReference Example 2 was prepared to have antibody concentration of 20mg/mL by replacing the medium with PBS6.5/EDTA by using the Commonprocedure C-2 and Common procedure B (as absorption coefficient at 280nm, 1.61 mLmg⁻¹cm⁻¹ was used). The solution (0.25 mL) was placed in a1.5 mL tube, charged with DMSO solution (0.0063 mL; which corresponds toabout 2.55 equivalents per antibody molecule) containing 27.6 mMsuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC,Thermo Fisher Scientific Inc.) at room temperature, and reacted at roomtemperature for 2 hours. This reaction solution was subjected topurification using the Common procedure D-2 to yield 0.7 mL of asolution containing about 5 mg of the SMCC-derivatized antibody.

Conjugation between antibody and drug linker: After adding DMSO (0.045mL) and a DMSO solution containing 10 mM of the compound obtained inProcess 1 (0.015 mL; which corresponds to about 2.4 equivalents perantibody molecule) to the above solution at room temperature, it wasstirred by using a tube rotator (MTR-103, manufactured by AS ONECorporation) for conjugating the drug linker to the antibody at roomtemperature for 16 hours.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) to yield 3.5 mLof a solution containing the titled antibody-drug conjugate. After that,the solution was concentrated by the Common procedure A.

Physicochemical characterization: By using the Common procedure E (asmolar absorption coefficient, ε_(A,280)=235300 (estimated calculationvalue), ε_(A,370)=0 (estimated calculation value), ε_(D,280)=5000(measured average value), and ε_(D,370)=19000 (measured average value)were used), the following characteristic values were obtained.

Antibody concentration: 3.85 mg/mL, antibody yield: 0.8 mg (16%), andaverage number of conjugated drug molecules (n) per antibody molecule:2.9.

Example 36 Antibody-Drug Conjugate (35)

Process 1: Antibody-Drug Conjugate (35)

SMCC derivatization of antibody: The M30-H1-L4P antibody produced inReference Example 2 was prepared to have antibody concentration of 20mg/mL by replacing the medium with PBS6.5/EDTA by using the Commonprocedure C-2 and Common procedure B (as absorption coefficient at 280nm, 1.61 mLmg⁻¹cm⁻¹ was used). The solution (0.25 mL) was placed in a1.5 mL tube, charged with DMSO solution (0.0125 mL; which corresponds toabout 5.1 equivalents per antibody molecule) containing 27.6 mMsuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC,Thermo Fisher Scientific Inc.) at room temperature, and reacted at roomtemperature for 2 hours. This reaction solution was subjected topurification using the Common procedure D-2 to yield 0.7 mL of asolution containing about 5 mg of the SMCC-derivatized antibody.

Conjugation between antibody and drug linker: After adding DMSO (0.03mL) and a DMSO solution containing 10 mM of the compound obtained inProcess 1 of Example 35 (0.03 mL; which corresponds to about 4.8equivalents per antibody molecule) to the above solution at roomtemperature, it was stirred by using a tube rotator (MTR-103,manufactured by AS ONE Corporation) for conjugating the drug linker tothe antibody at room temperature for 16 hours.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) to yield 3.5 mLof a solution containing the titled antibody-drug conjugate. After that,the solution was concentrated by the Common procedure A.

Physicochemical characterization: By using the Common procedure E (asmolar absorption coefficient, ε_(A,280)=235300 (estimated calculationvalue), ε_(A,370)=0 (estimated calculation value), ε_(D,280)=5000(measured average value), and ε_(D,370)=19000 (measured average value)were used), the following characteristic values were obtained.

Antibody concentration: 2.43 mg/mL, antibody yield: 0.5 mg (10%), andaverage number of conjugated drug molecules (n) per antibody molecule:4.2.

Example 37 Antibody-Drug Conjugate (36)

Process 1:N-{8-[(2,5-dioxopyrrolidin-1-yl)oxy]-8-oxooctanoyl}glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The compound (84.0 mg, 0.100 mmol) obtained in Process 2 of Example 2was reacted in the same manner as Process 3 of Example 2 by usingdi(N-succinimidyl) suberate instead of N-succinimidyl 6-maleimidehexanoate to yield the titled compound as a pale yellow solid (77.1 mg,71%).

¹H-NMR (DMSO-D₆) δ: 0.87 (3H, t, J=7.2 Hz), 1.21-1.38 (4H, m), 1.43-1.50(2H, m), 1.55-1.63 (2H, m), 1.68-1.76 (2H, m), 1.80-1.91 (2H, m),2.07-2.22 (6H, m), 2.40 (3H, s), 2.60-2.67 (2H, m), 2.76-2.84 (5H, m),2.97-3.22 (5H, m), 3.56-3.76 (6H, m), 4.40-4.50 (1H, m), 5.20 (2H, q,J=18.8 Hz), 5.37-5.48 (2H, m), 5.53-5.62 (1H, m), 6.54 (1H, s),7.15-7.28 (5H, m), 7.31 (1H, s), 7.71 (1H, t, J=5.5 Hz), 7.80 (1H, d,J=10.9 Hz), 8.04 (1H, t, J=5.9 Hz), 8.09 (1H, t, J=5.9 Hz), 8.14 (1H, d,J=7.8 Hz), 8.26 (1H, t, J=5.9 Hz), 8.47 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 1092 (M+H)⁺

Process 2: Antibody-Drug Conjugate (36)

Conjugation between antibody and drug linker: The M30-H1-L4P antibodyproduced in Reference Example 2 was prepared to have antibodyconcentration of 20 mg/mL by replacing the medium with PBS6.5/EDTA byusing the Common procedure C-2 and Common procedure B (as absorptioncoefficient at 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used). The solution (0.25 mL)was placed in a 1.5 mL tube, charged with a DMSO solution containing 10mM of the compound obtained in above Process 1 (0.025 mL; whichcorresponds to about 3.7 equivalents per antibody molecule) at roomtemperature, and stirred by using a tube rotator (MTR-103, manufacturedby AS ONE Corporation) for conjugating the drug linker to the antibodyat room temperature for 16 hours.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) to yield 3.5 mLof a solution containing the titled antibody-drug conjugate. After that,the solution was concentrated by the Common procedure A.

Physicochemical characterization: By using the Common procedure E (asmolar absorption coefficient, ε_(A,280)=235300 (estimated calculationvalue), ε_(A,370)=0 (estimated calculation value), ε_(D,280)=5000(measured average value), and ε_(D,370)=19000 (measured average value)were used), the following characteristic values were obtained.

Antibody concentration: 6.25 mg/mL, antibody yield: 1.3 mg (26%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.2.

Example 38 Antibody-Drug Conjugate (37)

Process 1: Antibody-Drug Conjugate (37)

Conjugation between antibody and drug linker: The M30-H1-L4P antibodyproduced in Reference Example 2 was prepared to have antibodyconcentration of 20 mg/mL by replacing the medium with PBS6.5/EDTA byusing the Common procedure C-2 and Common procedure B (as absorptioncoefficient at 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used). The solution (0.5 mL)was placed in a 1.5 mL tube, thereafter charged with a DMSO solutioncontaining DMSO (0.025 mL) and 10 mM of the compound obtained in Process1 of Example 37 (0.025 mL; which corresponds to about 7.4 equivalentsper antibody molecule) at room temperature, and stirred by using a tuberotator (MTR-103, manufactured by AS ONE Corporation) for conjugatingthe drug linker to the antibody at room temperature for 16 hours.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) to yield 3.5 mLof a solution containing the titled antibody-drug conjugate. After that,the solution was concentrated by the Common procedure A.

Physicochemical characterization: By using the Common procedure E (asmolar absorption coefficient, ε_(A,280)=235300 (estimated calculationvalue), ε_(A,370)=0 (estimated calculation value), ε_(D,280)=5000(measured average value), and ε_(D,370)=19000 (measured average value)were used), the following characteristic values were obtained.

Antibody concentration: 4.36 mg/mL, antibody yield: 0.9 mg (18%), andaverage number of conjugated drug molecules (n) per antibody molecule:4.1.

Example 39 Antibody-Drug Conjugate (38)

Process 1: Antibody-Drug Conjugate (38)

Conjugation between antibody and drug linker: The anti-CD30 antibodyproduced in Reference Example 3 was prepared to have antibodyconcentration of 10 mg/mL by replacing the medium with PBS6.5/EDTA byusing the Common procedure C-2 and Common procedure B (as absorptioncoefficient at 280 nm, 1.75 mLmg⁻¹cm⁻¹ was used). The solution (0.4 mL,4 mg of the antibody) was placed in a 1.5 mL tube, thereafter chargedwith DMSO (0.017 mL) and a DMSO solution containing 10 mM of thecompound obtained in Process 1 of Example 37 (0.023 mL; whichcorresponds to 9 equivalents per antibody molecule) at room temperature,and stirred by using a tube rotator (MTR-103, manufactured by AS ONECorporation) for conjugating the drug linker to the antibody at roomtemperature for 4 hours.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) to yield 2.5 mLof a solution containing the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure E (asmolar absorption coefficient, ε_(A,280)=270400 (estimated calculationvalue), ε_(A,370)=0 (estimated calculation value), ε_(D,280)=2670(measured value), and ε_(D,370)=15820 (measured value) were used), thefollowing characteristic values were obtained.

Antibody concentration: 0.55 mg/mL, antibody yield: 1.4 mg (34%), andaverage number of conjugated drug molecules (n) per antibody molecule:2.7.

Example 40 Antibody-Drug Conjugate (39)

Process 1: Antibody-Drug Conjugate (39)

Conjugation between antibody and drug linker: The anti-CD33 antibodyproduced in Reference Example 4 was prepared to have antibodyconcentration of 10 mg/mL by replacing the medium with PBS6.5/EDTA byusing the Common procedure C-2 and Common procedure B (as absorptioncoefficient at 280 nm, 1.66 mLmg⁻¹cm⁻¹ was used). The solution (0.4 mL,4 mg of the antibody) was placed in a 1.5 mL tube, thereafter chargedwith DMSO (0.017 mL) and a DMSO solution containing 10 mM of thecompound obtained in Process 1 of Example 37 (0.023 mL; whichcorresponds to 9 equivalents per antibody molecule) at room temperature,and stirred by using a tube rotator (MTR-103, manufactured by AS ONECorporation) for conjugating the drug linker to the antibody at roomtemperature for 4 hours.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) to yield 2.5 mLof a solution containing the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure E (asmolar absorption coefficient, ε_(A,280)=256400 (estimated calculationvalue), ε_(A,370)=0 (estimated calculation value), ε_(D,280)=2670(measured value), and ε_(D,370)=15820 (measured value) were used), thefollowing characteristic values were obtained. Antibody concentration:0.93 mg/mL, antibody yield: 2.3 mg (58%), and average number ofconjugated drug molecules (n) per antibody molecule: 4.0.

Example 41 Antibody-Drug Conjugate (40)

Process 1: Antibody-Drug Conjugate (40)

Conjugation between antibody and drug linker: The anti-CD70 antibodyproduced in Reference Example 5 was prepared to have antibodyconcentration of 10 mg/mL by replacing the medium with PBS6.5/EDTA byusing the Common procedure C-2 and Common procedure B (as absorptioncoefficient at 280 nm, 1.69 mLmg⁻¹cm⁻¹ was used). The solution (0.4 mL,4 mg of the antibody) was placed in a 1.5 mL tube, thereafter chargedwith DMSO (0.017 mL) and a DMSO solution containing 10 mM of thecompound obtained in Process 1 (0.023 mL; which corresponds to 9equivalents per antibody molecule) of Example 37 at room temperature,and stirred by using a tube rotator (MTR-103, manufactured by AS ONECorporation) for conjugating the drug linker to the antibody at roomtemperature for 4 hours.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) to yield 2.5 mLof a solution containing the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure E (asmolar absorption coefficient, ε_(A,280)=262400 (estimated calculationvalue), ε_(A,370)=0 (estimated calculation value), ε_(D,280)=2670(measured value), and ε_(D,370)=15820 (measured value) were used), thefollowing characteristic values were obtained.

Antibody concentration: 1.04 mg/mL, antibody yield: 2.6 mg (65%), andaverage number of conjugated drug molecules (n) per antibody molecule:4.1.

Example 422-(2-Aminoethoxy)-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]acetamide

Process 1: tert-Butyl[2-(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)ethyl]carbamate

Mesylate of the compound (4) (3.10 g, 5.47 mol) was reacted in the samemanner as Process 1 of Example 1 by using{2-[(tert-butoxycarbonyl)amino]ethoxy}acetic acid (J. Med. Chem., 1992,vol. 35, pp. 2928) (1.55 g, 6.01 mmol) instead of4-(tert-butoxycarbonylamino)butanoic acid to yield the titled compoundas a pale yellow solid (2.56 g, 73%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.3 Hz), 1.26 (9H, s),1.81-1.91 (2H, m), 2.13-2.22 (2H, m), 2.40 (3H, s), 3.08-3.26 (4H, m),3.43-3.53 (2H, m), 4.00 (1H, d, J=15.1 Hz), 4.05 (1H, d, J=15.1 Hz),5.14 (1H, d, J=18.7 Hz), 5.22 (1H, d, J=18.7 Hz), 5.40 (1H, d, J=16.6Hz), 5.44 (1H, d, J=16.6 Hz), 5.59-5.66 (1H, m), 6.53 (1H, s), 6.86 (1H,t, J=5.4 Hz), 7.31 (1H, s), 7.79 (1H, d, J=10.9 Hz), 8.49 (1H, d, J=9.1Hz). MS (APCI) m/z: 637 (M+H)⁺

Process 2:2-(2-Aminoethoxy)-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]acetamide

The compound (1.50 g, 2.36 mol) obtained in Process 1 above was reactedin the same manner as Process 2 of Example 1 to yieldtrifluorohydrochloride of the titled compound as a pale yellow solid(1.50 g, quantitative). This compound was confirmed in the tumor of acancer-bearing mouse that received the antibody-drug conjugate (41).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.5 Hz), 1.81-1.92 (2H, m),2.15-2.23 (2H, m), 2.41 (3H, s), 3.05 (2H, t, J=5.1 Hz), 3.15-3.23 (2H,m), 3.71 (2H, t, J=5.1 Hz), 4.10 (2H, s), 5.19 (1H, d, J=18.7 Hz), 5.24(1H, d, J=18.7 Hz), 5.43 (2H, s), 5.58-5.66 (1H, m), 6.55 (1H, s), 7.33(1H, s), 7.73-7.84 (4H, m), 8.55 (1H, d, J=9.1 Hz).

MS (APCI) m/z: 537 (M+H)⁺

Example 43 Antibody-Drug Conjugate (41)

Process 1:N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanyl-N-[2-(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)ethyl]glycinamide

The compound (554 mg, 0.85 mmol) of Example 42 was reacted in the samemanner as Process 1 of Example 2 to yield the titled compound (775 mg,95%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.85 (3H, t, J=7.3 Hz), 1.36 (9H, s),1.78-1.89 (2H, m), 2.13-2.22 (2H, m), 2.39 (3H, s), 2.71 (1H, dd,J=13.4, 9.8 Hz), 2.95 (1H, dd, J=13.4, 4.3 Hz), 3.09-3.23 (1H, m),3.23-3.32 (2H, m), 3.40-3.62 (8H, m), 3.73 (1H, dd, J=16.5, 5.5 Hz),4.03 (2H, s), 4.39-4.47 (1H, m), 5.17 (1H, d, J=18.9 Hz), 5.25 (1H, d,J=18.9 Hz), 5.41 (1H, d, J=16.8 Hz), 5.45 (1H, d, J=16.8 Hz), 5.57-5.64(1H, m), 6.54 (1H, s), 6.99 (1H, t, J=5.8 Hz), 7.13-7.26 (5H, m), 7.31(1H, s), 7.76-7.82 (2H, m), 7.90 (1H, t, J=5.2 Hz), 8.13 (1H, d, J=7.9Hz), 8.27 (1H, t, J=5.8 Hz), 8.49 (1H, d, J=8.5 Hz).

MS (APCI) m/z: 955 (M+H)⁺

Process 2:Glycylglycyl-L-phenylalanyl-N-[2-(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)ethyl]glycinamidetrifluoroacetate

The compound (630 mg, 0.659 mmol) obtained in Process 1 above wasreacted in the same manner as Process 2 of Example 2 to yield the titledcompound (588 mg, 92%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.3 Hz), 1.79-1.90 (2H, m),2.13-2.22 (2H, m), 2.39 (3H, s), 2.71 (1H, dd, J=13.4, 10.1 Hz), 2.99(1H, dd, J=13.4, 4.3 Hz), 3.09-3.23 (1H, m), 3.24-3.32 (3H, m),3.41-3.71 (7H, m), 3.86 (1H, dd, J=16.8, 5.8 Hz), 4.04 (2H, s), 4.52(1H, td, J=9.0, 4.1 Hz), 5.17 (1H, d, J=18.9 Hz), 5.25 (1H, d, J=18.9Hz), 5.41 (1H, d, J=16.5 Hz), 5.45 (1H, d, J=16.5 Hz), 5.56-5.65 (1H,m), 6.55 (1H, s), 7.13-7.26 (5H, m), 7.32 (1H, s), 7.80 (1H, d, J=11.0Hz), 7.87-8.01 (4H, m), 8.29-8.36 (2H, m), 8.46-8.55 (2H, m).

MS (APCI) m/z: 855 (M+H)⁺

Process 3:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[2-(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)ethyl]glycinamide

The compound (240 mg, 0.247 mmol) obtained in Process 2 above wasreacted in the same manner as Process 3 of Example 2 to yield the titledcompound (162 mg, 62%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.6 Hz), 1.13-1.22 (2H, m),1.40-1.51 (4H, m), 1.78-1.90 (2H, m), 2.09 (2H, t, J=7.6 Hz), 2.14-2.21(2H, m), 2.39 (3H, s), 2.74 (1H, dd, J=13.6, 9.7 Hz), 2.96 (1H, dd,J=13.6, 4.5 Hz), 3.08-3.24 (1H, m), 3.24-3.30 (1H, m), 3.33-3.40 (4H,m), 3.47-3.68 (7H, m), 3.72 (1H, dd, J=16.6, 5.7 Hz), 4.03 (2H, s), 4.42(1H, td, J=8.6, 4.2 Hz), 5.17 (1H, d, J=18.7 Hz), 5.25 (1H, d, J=18.7Hz), 5.40 (1H, d, J=17.2 Hz), 5.44 (1H, d, J=17.2 Hz), 5.57-5.64 (1H,m), 6.52 (1H, s), 6.99 (2H, s), 7.13-7.25 (5H, m), 7.31 (1H, s),7.74-7.81 (2H, m), 7.99 (1H, t, J=5.7 Hz), 8.03-8.11 (2H, m), 8.22 (1H,t, J=5.7 Hz), 8.47 (1H, d, J=9.1 Hz).

MS (APCI) m/z: 1048 (M+H)⁺

Process 4: Antibody-Drug Conjugate (41)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 3 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 2 of Example 29. Antibodyconcentration: 12.0 mg/mL, antibody yield: 8.4 mg (67%), and averagenumber of conjugated drug molecules (n) per antibody molecule: 3.5.

Example 44 Antibody-Drug Conjugate (42)

Process 1: Antibody-Drug Conjugate (42)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 3 of Example 43, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 5.

Antibody concentration: 0.83 mg/mL, antibody yield: 4.98 mg (40%), andaverage number of conjugated drug molecules (n) per antibody molecule:7.2.

Example 45 Antibody-Drug Conjugate (43)

Process 1: Antibody-Drug Conjugate (43)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 3 of Example 43, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 4.

Antibody concentration: 1.06 mg/mL, antibody yield: 6.36 mg (51%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.3.

Example 46 Antibody-Drug Conjugate (44)

Almost the whole amounts of the antibody-drug conjugates of Examples 44and 45 were mixed and the solution was concentrated by the Commonprocedure A to yield the titled antibody-drug conjugate.

Antibody concentration: 10.0 mg/mL, antibody yield: 10.21 mg, andaverage number of conjugated drug molecules (n) per antibody molecule:6.6.

Example 47 Antibody-Drug Conjugate (45)

Process 1: Antibody-Drug Conjugate (45)

Reduction of the antibody: The M30-H1-L4 antibody produced in ReferenceExample 1 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (1.25 mL, 12.5 mg of the antibody)was placed in a 1.5 mL tube and charged with an aqueous solution of 10mM TCEP (Tokyo Chemical Industry Co., Ltd.) (0.0287 mL; 3.4 equivalentsper antibody molecule) and an aqueous solution of 1 M dipotassiumhydrogen phosphate (Nacalai Tesque, Inc.; 0.0625 mL). After confirmingthat the solution had pH of 7.4±0.1, the disulfide bond at hinge part inthe antibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (0.0267 mL) and a dimethyl sulfoxide solution containing 10 mMof the compound obtained in Process 3 of Example 43 (0.0439 mL; 5.2equivalents per antibody molecule) to the above solution at roomtemperature, it was incubated for conjugating the drug linker to theantibody in a water bath at 15° C. for 1 hour. Next, an aqueous solution(0.0066 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was added thereto andincubated to terminate the raction of drug linker at room temperaturefor another 20 minutes. Purification: The above solution was subjectedto purification using the Common procedure D-1 (ABS was used as buffersolution) described in Production method to yield 6 mL of a solutioncontaining the titled antibody-drug conjugate. After that, the solutionwas concentrated by the Common procedure A.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5193 (measured value), and ε_(D,370)=20347(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 10.0 mg/mL, antibody yield: 9.3 mg (74%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.7.

Example 48 Antibody-Drug Conjugate (46)

Process 1: Antibody-Drug Conjugate (46)

Reduction of the antibody: The M30-H1-L4 antibody produced in ReferenceExample 1 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (1.25 mL, 12.5 mg of the antibody)was placed in a 1.5 mL tube and charged with an aqueous solution of 10mM TCEP (Tokyo Chemical Industry Co., Ltd.) (0.0439 mL; 5.2 equivalentsper antibody molecule) (0.0287 mL; 3.4 equivalents per antibodymolecule) and an aqueous solution of 1 M dipotassium hydrogen phosphate(Nacalai Tesque, Inc.; 0.0625 mL). After confirming that the solutionhad pH of 7.4±0.1, the disulfide bond at hinge part in the antibody wasreduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution (0.0726 mL; 8.6 equivalents per antibody molecule)containing 10 mM of the compound obtained in Process 3 of Example 43 tothe above solution at room temperature, it was incubated for conjugatingthe drug linker to the antibody in a water bath at 15° C. for 1 hour.Next, an aqueous solution (0.011 mL) of 100 mM NAC (Sigma-Aldrich Co.LLC) was added thereto and incubated to terminate the raction of druglinker at room temperature for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate. After that, the solution was concentrated bythe Common procedure A.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5193 (measured value), and ε_(D,370)=20347(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 10.0 mg/mL, antibody yield: 7.8 mg (62%), andaverage number of conjugated drug molecules (n) per antibody molecule:5.2.

Example 49N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

Process 1: tert-Butyl(3-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-3-oxopropyl)carbamate

Mesylate of the compound (4) (500 mg, 0.941 mmol) was reacted in thesame manner as Process 1 of Example 1 by usingN-(tert-butoxycarbonyl)-β-alanine instead of4-(tert-butoxycarbonylamino)butanoic acid to yield the titled compoundas a yellow-brown solid (616 mg, quantitative).

¹H-NMR (400 MHz, DMSO-d₅) δ: 0.87 (3H, t, J=7.2 Hz), 1.29 (9H, s), 1.86(2H, dt, J=15.1, 7.3 Hz), 2.04-2.22 (2H, m), 2.31 (2H, t, J=6.8 Hz),2.40 (3H, s), 3.10-3.26 (4H, m), 5.15 (1H, d, J=18.8 Hz), 5.26 (1H, d,J=19.2 Hz), 5.42 (2H, dd, J=18.8, 16.4 Hz), 5.57 (1H, dt, J=8.5, 4.2Hz), 6.53 (1H, s), 6.78 (1H, t, J=5.5 Hz), 7.30 (1H, s), 7.80 (1H, d,J=11.0 Hz), 8.46 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 607 (M+H)⁺

Process 2:N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

The compound obtained in Process 1 above was reacted in the same manneras Process 2 of Example 1 to yield trifluoroacetate of the titledcompound as a yellow solid (499 mg, 86%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.2 Hz), 1.86 (2H, dquin,J=14.6, 7.2, 7.2, 7.2, 7.2 Hz), 2.06-2.27 (1H, m), 2.41 (3H, s),2.46-2.57 (2H, m), 3.08 (2H, t, J=6.8 Hz), 3.14-3.24 (2H, m), 5.22 (1H,d, J=18.8 Hz), 5.29 (1H, d, J=18.8 Hz), 5.43 (2H, s), 5.58 (1H, dt,J=8.5, 4.5 Hz), 6.55 (1H, s), 7.32 (1H, s), 7.74 (3H, brs), 7.82 (1H, d,J=11.0 Hz), 8.67 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 507 (M+H)⁺

Example 50 Antibody-Drug Conjugate (47)

Process 1:N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

The compound (484 mg, 0.780 mmol) of Example 49 was reacted in the samemanner as Process 1 of Example 2 to yield the titled compound as a paleyellow solid (626 mg, 87%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.27-1.42 (9H, m),1.77-1.93 (2H, m), 2.06-2.22 (2H, m), 2.36 (2H, t, J=7.2 Hz), 2.40 (3H,d, J=1.6 Hz), 2.44-2.54 (2H, m), 2.76 (1H, dd, J=14.5, 10.2 Hz), 3.02(1H, dd, J=13.9, 4.5 Hz), 3.12-3.22 (2H, m), 3.52 (6H, d, J=6.3 Hz),4.42-4.54 (1H, m), 5.19 (1H, d, J=19.2 Hz), 5.26 (1H, d, J=18.4 Hz),5.42 (1H, dd, J=18.4, 16.4 Hz), 5.57 (1H, dt, J=8.7, 4.4 Hz), 6.53 (1H,s), 6.98 (1H, t, J=5.9 Hz), 7.14-7.28 (5H, m), 7.31 (1H, s), 7.77-7.84(1H, m), 7.91 (1H, t, J=5.5 Hz), 8.16 (1H, d, J=7.8 Hz), 8.27 (1H, t,J=5.1 Hz), 8.52 (1H, d, J=9.0 Hz).

Process 2:Glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamidetrifluoroacetate

The compound (624 mg, 0.675 mmol) obtained in Process 1 above wasreacted in the same manner as Process 2 of Example 2 to yield the titledcompound as a yellow solid (626 mg, 92%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.86 (2H, tt,J=14.5, 7.2 Hz), 2.07-2.22 (2H, m), 2.36 (2H, t, J=7.2 Hz), 2.40 (3H,s), 2.44-2.54 (2H, m), 2.75 (1H, dd, J=13.7, 9.8 Hz), 3.04 (1H, dd,J=13.7, 4.3 Hz), 3.12-3.22 (2H, m), 3.58 (2H, d, J=4.7 Hz), 3.69 (3H,td, J=11.2, 5.7 Hz), 3.87 (1H, dd, J=17.0, 5.7 Hz), 4.54 (1H, m, J=17.8,4.5 Hz), 5.19 (1H, d, J=19.2 Hz), 5.26 (1H, d, J=18.8 Hz), 5.43 (2H, s),5.51-5.60 (1H, m), 6.55 (1H, s), 7.14-7.29 (5H, m), 7.32 (1H, s), 7.81(1H, d, J=10.9 Hz), 7.88 (1H, t, J=5.7 Hz), 7.97 (3H, brs), 8.29-8.38(2H, m), 8.50 (1H, t, J=5.7 Hz), 8.55 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 825 (M+H)⁺

Process 3:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 above wasreacted in the same manner as Process 3 of Example 2 to yield the titledcompound as a solid (14.0 mg, 21%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.2 Hz), 1.12-1.22 (2H, m),1.39-1.51 (4H, m), 1.79-1.91 (2H, m), 2.02-2.20 (2H, m), 2.07 (2H, t,J=7.4 Hz), 2.30-2.42 (4H, m), 2.40 (3H, s), 2.78 (1H, dd, J=14.1, 9.4Hz), 3.02 (1H, dd, J=14.7, 4.9 Hz), 3.12-3.21 (2H, m), 3.26-3.42 (2H,m), 3.50-3.80 (6H, m), 4.40-4.51 (1H, m), 5.19 (1H, d, J=19.6 Hz), 5.26(1H, d, J=19.2 Hz), 5.42 (2H, brs), 5.51-5.62 (1H, m), 6.53 (1H, s),6.99 (2H, s), 7.13-7.28 (5H, m), 7.31 (1H, s), 7.74-7.84 (2H, m), 8.01(1H, t, J=5.3 Hz), 8.06 (1H, t, J=5.7 Hz), 8.14 (1H, d, J=8.2 Hz), 8.25(1H, t, J=5.7 Hz), 8.53 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 1018 (M+H)⁺

Process 4: Antibody-Drug Conjugate (47)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 3 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 4 of Example 2. Antibodyconcentration: 12.27 mg/mL, antibody yield: 8.6 mg (69%), and averagenumber of conjugated drug molecules (n) per antibody molecule: 3.4.

Example 51 Antibody-Drug Conjugate (48)

Process 1:N-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 of Example 50was reacted in the same manner as Process 3 of Example 2 by usingN-succinimidyl 3-maleimide propionate instead of N-succinimidyl6-maleimide hexanoate to yield the titled compound as a pale yellowsolid (36.0 mg, 57%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.4 Hz), 1.85 (2H, dt,J=14.4, 7.5 Hz), 2.05-2.22 (2H, m), 2.40 (3H, s), 2.30-2.44 (5H, m),2.73-2.84 (1H, m), 3.02 (1H, dd, J=13.9, 4.5 Hz), 3.17 (3H, d, J=5.1Hz), 3.26-3.40 (2H, m), 3.41-3.81 (6H, m), 4.40-4.51 (1H, m), 5.19 (1H,d, J=19.2 Hz), 5.26 (1H, d, J=18.8 Hz), 5.42 (2H, brs), 5.52-5.61 (1H,m), 6.53 (1H, s), 6.99 (2H, s), 7.13-7.28 (5H, m), 7.31 (1H, s), 7.80(2H, d, J=10.2 Hz), 8.03 (1H, t, J=5.5 Hz), 8.12 (1H, d, J=8.2 Hz),8.20-8.31 (2H, m), 8.52 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 976 (M+H)⁺

Process 2: Antibody-Drug Conjugate (48)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 1 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 4 of Example 2. Antibodyconcentration: 11.59 mg/mL, antibody yield: 8.1 mg (65%), and averagenumber of conjugated drug molecules (n) per antibody molecule: 3.7.

Example 52 Antibody-Drug Conjugate (49)

Process 1:N-{3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino})ethoxy]propanoyl}glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 of Example 50was reacted in the same manner as Process 3 of Example 2 by usingN-succinimidyl3-(2-(2-(3-maleinimidepropanamide)ethoxy)ethoxy)propanoate instead ofN-succinimidyl 6-maleimide hexanoate to yield the titled compound as asolid (23.0 mg, 31%).

¹H-NMR (400 MHz, DMSO-d₅) δ: 0.86 (3H, t, J=7.4 Hz), 1.77-1.92 (2H, m),2.07-2.21 (2H, m), 2.27-2.42 (6H, m), 2.40 (3H, s), 2.74-2.84 (1H, m),2.97-3.06 (1H, m), 3.09-3.21 (4H, m), 3.25-3.39 (6H, m), 3.45 (4H, s),3.50-3.80 (8H, m), 4.41-4.51 (1H, m), 5.19 (1H, d, J=18.4 Hz), 5.26 (1H,m, J=18.4 Hz), 5.42 (2H, brs), 5.51-5.61 (1H, m), 6.54 (1H, s), 7.00(2H, s), 7.13-7.28 (5H, m), 7.31 (1H, s), 7.74-7.87 (2H, m), 7.93-8.07(2H, m), 8.09-8.21 (2H, m), 8.26 (1H, brs), 8.54 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 1135 (M+H)⁺

Process 2: Antibody-Drug Conjugate (49)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 1 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 2 of Example 29. Antibodyconcentration: 14.50 mg/mL, antibody yield: 10.2 mg (82%), and averagenumber of conjugated drug molecules (n) per antibody molecule: 3.8.

Example 53 Antibody-Drug Conjugate (50)

Process 1:N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 of Example 50was reacted in the same manner as Process 3 of Example 2 by usingN-succinimidyl1-maleinimide-3-oxo-7,10,13,16-tetraoxa-4-azanonadecanoate instead ofN-succinimidyl 6-maleimide hexanoate to yield the titled compound as asolid (23.0 mg, 29%).

¹H-NMR (400 MHz, DMSO-d₅) δ: 0.86 (3H, t, J=7.0 Hz), 1.85 (2H, tt,J=14.6, 7.1 Hz), 2.06-2.22 (2H, m), 2.40 (3H, s), 2.28-2.43 (6H, m),2.78 (1H, dd, J=13.7, 9.4 Hz), 3.02 (1H, dd, J=14.1, 3.9 Hz), 3.09-3.22(4H, m), 3.27-3.41 (4H, m), 3.47 (12H, d, J=8.6 Hz), 3.53-3.81 (10H, m),4.41-4.51 (1H, m), 5.19 (1H, d, J=19.2 Hz), 5.26 (1H, d, J=18.8 Hz),5.42 (2H, brs), 5.53-5.61 (1H, m), 6.54 (1H, s), 7.00 (2H, s), 7.12-7.29(5H, m), 7.31 (1H, s), 7.74-7.85 (2H, m), 8.03 (2H, d, J=6.6 Hz),8.11-8.21 (2H, m), 8.27 (1H, t, J=5.9 Hz), 8.54 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 1224 (M+H)⁺

Process 2: Antibody-Drug Conjugate (50)

By using the M30-H1-L4P antibody produced in Reference Example 2 and thecompound obtained in Process 1 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 4 of Example 2. Antibodyconcentration: 13.47 mg/mL, antibody yield: 9.4 mg (75%), and averagenumber of conjugated drug molecules (n) per antibody molecule: 3.1.

Example 54 Antibody-Drug Conjugate (51)

Process 1: tert-Butyl(6-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-6-oxohexyl)carbamate

Mesylate of the compound (4) (0.500 g, 0.882 mmol) was reacted in thesame manner as Process 1 of Example by using6-(tert-butoxycarbonylamino)hexanoic acid instead of4-(tert-butoxycarbonylamino)butanoic acid to yield the titled compound(0.620 g, quantitative).

¹H-NMR (DMSO-d₆) δ: 0.83 (3H, t, J=7.8 Hz), 1.14-1.28 (2H, m), 1.31 (9H,s), 1.47-1.61 (2H, m), 1.75-1.89 (2H, m), 2.04-2.17 (4H, m), 2.35 (3H,s), 2.81-2.88 (2H, m), 3.09-3.16 (2H, m), 5.10 (1H, d, J=19.4 Hz), 5.16(1H, d, J=19.4 Hz), 5.39 (2H, s), 5.48-5.55 (1H, m), 6.50 (1H, s),6.73-6.78 (1H, m), 7.26 (1H, s), 7.74 (1H, d, J=10.9 Hz), 8.39 (1H, d,J=9.0 Hz).

Process 2:6-Amino-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]hexanamidetrifluoroacetate

The compound (0.397 g, 0.611 mmol) obtained in Process 1 above wasreacted in the same manner as Process 2 of Example 1 to yield the titledcompound (0.342 g, 84%).

¹H-NMR (DMSO-d₆) δ: 0.88 (3H, t, J=7.2 Hz), 1.31-1.41 (2H, m), 1.52-1.70(4H, m), 1.80-1.94 (2H, m), 2.05-2.18 (2H, m), 2.21 (2H, t, J=7.4 Hz),2.40 (3H, s), 2.81 (2H, t, J=7.4 Hz), 3.10-3.25 (2H, m), 3.33 (2H, brs),5.18 (1H, d, J=19.8 Hz), 5.22 (1H, d, J=19.8 Hz), 5.41 (2H, d, J=16.6Hz), 5.45 (2H, d, J=16.6 Hz), 5.53-5.60 (1H, m), 6.55 (1H, s), 7.32 (1H,s), 7.80 (1H, d, J=10.9 Hz), 8.49 (1H, d, J=9.2 Hz).

Process 3:N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanyl-N-(6-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-6-oxohexyl)glycinamide

The compound (0.170 g, 0.516 mmol) obtained in Process 2 above wasreacted in the same manner as Process 1 of Example 2 to yield the titledcompound (0.225 g, 91%).

¹H-NMR (DMSO-d₆) δ: 0.88 (3H, t, J=7.4 Hz), 1.43-1.70 (6H, m), 1.87 (2H,td, J=15.0, 7.4 Hz), 2.10-2.22 (3H, m), 2.28-2.37 (1H, m), 2.42 (3H, s),2.78-2.85 (1H, m), 3.01-3.10 (3H, m), 3.15-3.22 (2H, m), 3.54-3.61 (5H,m), 3.62-3.69 (1H, m), 4.44-4.53 (1H, m), 5.17 (1H, d, J=19.2 Hz), 5.25(1H, d, J=19.2 Hz), 5.45 (2H, s), 5.54-5.61 (1H, m), 6.55 (1H, s), 7.02(1H, t, J=6.1 Hz), 7.11-7.28 (5H, m), 7.33 (1H, s), 7.63-7.69 (1H, m),7.82 (1H, d, J=11.0 Hz), 7.90-7.96 (1H, m), 8.17 (1H, d, J=7.8 Hz), 8.28(1H, t, J=5.5 Hz), 8.46 (1H, d, J=9.0 Hz).

Process 4:Glycylglycyl-L-phenylalanyl-N-(6-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-6-oxohexyl)glycinamide

The compound (0.105 g, 0.108 mmol) obtained in Process 3 above wasreacted in the same manner as Process 2 of Example 2 to yield the titledcompound (0.068 mg, 65%).

¹H-NMR (DMSO-d₆) δ: 0.89 (3H, t, J=7.4 Hz), 1.15-1.67 (6H, m), 1.79-1.97(2H, m), 2.08-2.24 (4H, m), 2.42 (3H, s), 2.76-2.82 (1H, m), 3.00-3.10(5H, m), 3.19 (1H, s), 3.50-3.63 (2H, m), 3.64-3.76 (3H, m), 3.84-3.92(1H, m), 4.51-4.59 (1H, m), 5.17 (1H, d, J=19.4 Hz), 5.24 (1H, d, J=19.4Hz), 5.44 (2H, s), 5.53-5.61 (1H, m), 6.55 (1H, brs), 7.15-7.29 (5H, m),7.33 (1H, s), 7.72-7.78 (1H, m), 7.82 (1H, d, J=11.0 Hz), 7.96-8.08 (2H,m), 8.30-8.38 (2H, m), 8.46-8.56 (2H, m).

Process 5:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-(6-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-6-oxohexyl)glycinamide

The compound (58 mg, 0.060 mmol) obtained in Process 4 above was reactedin the same manner as Process 3 of Example 2 to yield the titledcompound (39 mg, 62%).

¹H-NMR (CD₃OD) δ: 0.99 (3H, t, J=7.4 Hz), 1.27 (2H, td, J=11.6, 6.1 Hz),1.38-1.44 (2H, m), 1.50-1.63 (6H, m), 1.65-1.80 (2H, m), 1.89-1.98 (2H,m), 2.17-2.25 (3H, m), 2.26-2.36 (3H, m), 2.40 (3H, s), 2.95 (1H, dd,J=14.3, 9.2 Hz), 3.12 (1H, dd, J=13.7, 5.7 Hz), 3.15-3.25 (4H, m), 3.44(2H, t, J=7.2 Hz), 3.65 (1H, d, J=17.2 Hz), 3.76 (1H, d, J=17.2 Hz),3.79-3.86 (4H, m), 4.43 (1H, dd, J=8.9, 6.0 Hz), 5.10 (1H, d, J=18.9Hz), 5.25 (1H, d, J=18.9 Hz), 5.35 (1H, d, J=16.6 Hz), 5.56 (1H, d,J=16.0 Hz), 5.60-5.64 (1H, m), 6.76 (2H, s), 7.12-7.24 (6H, m), 7.58(1H, s), 7.60 (1H, d, J=10.9 Hz), 7.68 (1H, t, J=5.7 Hz).

MS (ESI) m/z: 1060 (M+H)⁺

Process 6: Antibody-Drug Conjugate (51)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 was used) and Common procedure C-1 described inProduction method 1. The solution (1.0 mL) was collected into a 1.5 mLtube and charged with an aqueous solution of 10 mM TCEP (Tokyo ChemicalIndustry Co., Ltd.) (0.0147 mL; 2.3 equivalents per antibody molecule)and an aqueous solution of 1 M dipotassium hydrogen phosphate (NacalaiTesque, Inc.; 0. 050 mL). After confirming that the solution had pH of7.4±0.1, the disulfide bond at hinge part in the antibody was reduced byincubating at 37° C. for 1 hour. Conjugation between antibody and druglinker: After incubating the solution at 22° C. for 10 minutes, adimethyl sulfoxide solution containing 10 mM of the compound obtained inabove Process 5 (0.0295 mL; 4.6 equivalents per antibody molecule) wasadded thereto and incubated for conjugating the drug linker to theantibody at 22° C. for 40 minutes. Next, an aqueous solution (0.00590mL; 9.2 equivalents per antibody molecule) of 100 mM NAC (Sigma-AldrichCo. LLC) was added thereto and incubated to terminate the raction ofdrug linker at 22° C. for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (PBS7.4 was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 0.97 mg/mL, antibody yield: 5.82 mg (58%), andaverage number of conjugated drug molecules (n) per antibody molecule:1.7.

Example 55 Antibody-Drug Conjugate (52)

Process 1: Antibody-Drug Conjugate (52)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 was used) and Common procedure C-1 described inProduction method 1. The solution (1.0 mL) was collected into a 1.5 mLtube and charged with an aqueous solution of 10 mM TCEP (Tokyo ChemicalIndustry Co., Ltd.) (0.0295 mL; 4.6 equivalents per antibody molecule)and an aqueous solution of 1 M dipotassium hydrogen phosphate (NacalaiTesque, Inc.; 0. 050 mL). After confirming that the solution had pH of7.4±0.1, the disulfide bond at hinge part in the antibody was reduced byincubating at 37° C. for 1 hour. Conjugation between antibody and druglinker: After incubating the above solution at 22° C. for 10 minutes, adimethyl sulfoxide solution containing 10 mM of the compound obtained inProcess 5 of Example 54 (0.0590 mL; 9.2 equivalents per antibodymolecule) was added thereto and incubated for conjugating the druglinker to the antibody at 22° C. for 40 minutes. Next, an aqueoussolution (0.0118 mL; 18.4 equivalents per antibody molecule) of 100 mMNAC (Sigma-Aldrich Co. LLC) was added thereto and incubated to terminatethe raction of drug linker at 22° C. for another 20 minutes.Purification: The above solution was subjected to purification using theCommon procedure D-1 (PBS7.4 was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 0.94 mg/mL, antibody yield: 5.64 mg (56%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.1.

Example 56 Antibody-Drug Conjugate (53)

Process 1: Antibody-Drug Conjugate (53)

Reduction of the antibody: The M30-H1-L4 antibody produced in ReferenceExample 1 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 was used) and Common procedure C-1 described inProduction method 1. The solution (1.0 mL) was collected into a 1.5 mLtube and charged with an aqueous solution of 10 mM TCEP (Tokyo ChemicalIndustry Co., Ltd.) (0.0147 mL; 2.3 equivalents per antibody molecule)and an aqueous solution of 1 M dipotassium hydrogen phosphate (NacalaiTesque, Inc.; 0. 050 mL). After confirming that the solution had pH of7.4±0.1, the disulfide bond at hinge part in the antibody was reduced byincubating at 37° C. for 1 hour. Conjugation between antibody and druglinker: After incubating the above solution at 22° C. for 10 minutes, adimethyl sulfoxide solution containing 10 mM of the compound obtained inProcess 5 of Example 54 (0.0295 mL; 4.6 equivalents per antibodymolecule) was added thereto and incubated for conjugating the druglinker to the antibody at 22° C. for 40 minutes. Next, an aqueoussolution (0.00590 mL; 9.2 equivalents per antibody molecule) of 100 mMNAC (Sigma-Aldrich Co. LLC) was added thereto and incubated to terminatethe raction of drug linker at 22° C. for another 20 minutes.Purification: The above solution was subjected to purification using theCommon procedure D-1 (PBS7.4 was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 1.22 mg/mL, antibody yield: 7.32 mg (73%), andaverage number of conjugated drug molecules (n) per antibody molecule:1.5.

Example 57 Antibody-Drug Conjugate (54)

Process 1: Antibody-Drug Conjugate (54)

Reduction of the antibody: The M30-H1-L4 antibody produced in ReferenceExample 1 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 was used) and Common procedure C-1 described inProduction method 1. The solution (1.0 mL) was collected into a 1.5 mLtube and charged with an aqueous solution of 10 mM TCEP (Tokyo ChemicalIndustry Co., Ltd.) (0.0295 mL; 4.6 equivalents per antibody molecule)and an aqueous solution of 1 M dipotassium hydrogen phosphate (NacalaiTesque, Inc.; 0. 050 mL). After confirming that the solution had pH of7.4±0.1, the disulfide bond at hinge part in the antibody was reduced byincubating at 37° C. for 1 hour. Conjugation between antibody and druglinker: After incubating the above solution at 22° C. for 10 minutes, adimethyl sulfoxide solution containing 10 mM of the compound obtained inProcess 5 of Example 54 (0.0590 mL; 9.2 equivalents per antibodymolecule) was added thereto and incubated for conjugating the druglinker to the antibody at 22° C. for 40 minutes. Next, an aqueoussolution (0.0118 mL; 18.4 equivalents per antibody molecule) of 100 mMNAC (Sigma-Aldrich Co. LLC) was added thereto and incubated to terminatethe raction of drug linker at 22° C. for another 20 minutes.Purification: The above solution was subjected to purification using theCommon procedure D-1 (PBS7.4 was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 1.06 mg/mL, antibody yield: 6.36 mg (64%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.0.

Example 58 Antibody-Drug Conjugate (55)

Process 1: ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methylacetate

To a mixture containing N-9-fluorenylmethoxycarbonylglycylglycine (4.33g, 12.2 mmol), tetrahydrofuran (120 ml), and toluene (40.0 ml), pyridine(1.16 ml, 14.7 mmol) and lead tetraacetate (6.84 g, 14.7 mmol) wereadded and refluxed under heating for 5 hours. After the reactionsolution was cooled to room temperature, the insolubles were removed byfiltration through Celite, and concentrated under reduced pressure. Theresidues obtained were dissolved in ethyl acetate and washed with waterand saturated brine, and then the organic layer was dried over anhydrousmagnesium sulfate. After the solvent was removed under reduced pressure,the residues obtained were purified by silica gel column chromatography[hexane:ethyl acetate=9:1 (v/v)-ethyl acetate] to yield the titledcompound as a colorless solid (3.00 g, 67%).

¹H-NMR (400 MHz, CDCl₃) δ: 2.07 (3H, s), 3.90 (2H, d, J=5.1 Hz), 4.23(1H, t, J=7.0 Hz), 4.46 (2H, d, J=6.6 Hz), 5.26 (2H, d, J=7.0 Hz), 5.32(1H, brs), 6.96 (1H, brs), 7.32 (2H, t, J=7.3 Hz), 7.41 (2H, t, J=7.3Hz), 7.59 (2H, d, J=7.3 Hz), 7.77 (2H, d, J=7.3 Hz).

Process 2: Benzyl [({N-[(9H-fluoren-9-ylmethoxy) carbonyl]glycyl}amino)methoxy]acetate

To a tetrahydrofuran (40.0 mL) solution of the compound (3.68 g, 10.0mmol) obtained in Process 1 above and benzyl glycolate (4.99 g, 30.0mmol), potassium tert-butoxide (2.24 g, 20.0 mmol) was added at 0° C.and stirred at room temperature for 15 minutes. The reaction solutionwas charged with ethyl acetate and water at 0° C. and extracted withethyl acetate and chloroform. The obtained organic layer was dried oversodium sulfate and filtered. The solvent was removed under reducedpressure. The residues obtained were dissolved in dioxane (40.0 mL) andwater (10.0 mL), charged with sodium hydrogen carbonate (1.01 g, 12.0mmol) and 9-fluorenylmethyl chloroformate (2.59 g, 10.0 mmol), andstirred at room temperature for 2 hours. The reaction solution wascharged with water and extracted with ethyl acetate. The obtainedorganic layer was dried over sodium sulfate and filtered. The solventwas removed under reduced pressure and the residues obtained werepurified by silica gel column chromatography [hexane:ethyl acetate=100:0(v/v)-0:100] to yield the titled compound in colorless oily substance(1.88 g, 40%).

¹H-NMR (400 MHz, CDCl₃) δ: 3.84 (2H, d, J=5.5 Hz), 4.24 (3H, t, J=6.5Hz), 4.49 (2H, d, J=6.7 Hz), 4.88 (2H, d, J=6.7 Hz), 5.15-5.27 (1H, m),5.19 (2H, s), 6.74 (1H, brs), 7.31-7.39 (7H, m), 7.43 (2H, t, J=7.4 Hz),7.61 (2H, d, J=7.4 Hz), 7.79 (2H, d, J=7.4 Hz).

Process 3: [({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetic acid

The compound (1.88 g, 3.96 mmol) obtained in Process 2 above wasdissolved in ethanol (40.0 mL) and ethyl acetate (20.0 ml). After addingpalladium carbon catalyst (376 mg), it was stirred under hydrogenatmosphere at room temperature for 2 hours. The insolubles were removedby filtration through Celite, and the solvent was removed under reducedpressure to yield the titled compound as a colorless solid (1.52 g,quantitative).

¹H-NMR (400 MHz, DMSO-d₆) δ: 3.62 (2H, d, J=6.3 Hz), 3.97 (2H, s),4.18-4.32 (3H, m), 4.60 (2H, d, J=6.7 Hz), 7.29-7.46 (4H, m), 7.58 (1H,t, J=5.9 Hz), 7.72 (2H, d, J=7.4 Hz), 7.90 (2H, d, J=7.4 Hz), 8.71 (1H,t, J=6.5 Hz).

Process 4:9H-Fluoren-9-ylmethyl(2-{[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]amino}-2-oxoethyl)carbamate

Under ice cooling, to an N,N-dimethylformamide (10.0 mL) solution ofmesylate of the compound (4) (0.283 g, 0.533 mmol), N-hydroxysuccinimide(61.4 mg, 0.533 mmol), and the compound (0.205 g, 0.533 mmol) obtainedin Process 3 above, N,N-diisopropylethylamine (92.9 μL, 0.533 mmol) andN,N′-dicyclohexylcarbodiimide (0.143 g, 0.693 mmol) were added andstirred at room temperature for 3 days. The solvent was removed underreduced pressure and the residues obtained were purified by silica gelcolumn chromatography [chloroform-partitioned organic layer ofchloroform:methanol:water=7:3:1 (v/v/v)] to yield the titled compound asa pale brown solid (0.352 g, 82%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.81 (3H, t, J=7.4 Hz), 1.73-1.87 (2H, m),2.06-2.20 (2H, m), 2.34 (3H, s), 3.01-3.23 (2H, m), 3.58 (2H, d, J=6.7Hz), 3.98 (2H, s), 4.13-4.25 (3H, m), 4.60 (2H, d, J=6.7 Hz), 5.09-5.22(2H, m), 5.32-5.42 (2H, m), 5.50-5.59 (1H, m), 6.49 (1H, s), 7.24-7.30(3H, m), 7.36 (2H, t, J=7.4 Hz), 7.53 (1H, t, J=6.3 Hz), 7.66 (2H, d,J=7.4 Hz), 7.75 (1H, d, J=11.0 Hz), 7.84 (2H, d, J=7.4 Hz), 8.47 (1H, d,J=8.6 Hz), 8.77 (1H, t, J=6.7 Hz).

MS (ESI) m/z: 802 (M+H)⁺

Process 5:N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

To an N,N-dimethylformamide (11.0 mL) solution of the compound (0.881 g,1.10 mmol) obtained in Process 4 above, piperidine (1.1 mL) was addedand stirred at room temperature for 2 hours. The solvent was removedunder reduced pressure to yield a mixture containing the titledcompound. The mixture was used for the next reaction without furtherpurification.

Process 6:N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

Under ice cooling, to an N,N-dimethylformamide (50.0 mL) solution of themixture (0.439 mmol) obtained in Process 5 above, N-hydroxysuccinimide(0.101 g, 0.878 mmol), andN-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanine (thecompound described in Japanese Patent Laid-Open No. 2002-60351) (0.440g, 0.878 mmol), N,N′-dicyclohexylcarbodiimide (0.181 g, 0.878 mmol) wasadded and stirred at room temperature for 4 days. The solvent wasremoved under reduced pressure and the residues obtained were purifiedby silica gel column chromatography [chloroform-chloroform:methanol=: 1(v/v)] to yield the titled compound as a pale orange solid (0.269 g,58%).

MS (ESI) m/z: 1063 (M+H)⁺

Process 7:Glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

To an N,N-dimethylformamide (4.00 mL) solution of the compound (0.269 g,0.253 mmol) obtained in Process 6 above, piperidine (0.251 mL, 2.53mmol) was added and stirred at room temperature for 2 hours. The solventwas removed under reduced pressure to yield a mixture containing thetitled compound. The mixture was used for the next reaction withoutfurther purification.

Process 8:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

To an N,N-dimethylformamide (10.0 mL) solution of the compound (0.253mmol) obtained in Process 7 above, N-succinimidyl 6-maleimide hexanoate(0.156 g, 0.506 mmol) was added and stirred at room temperature for 3days. The solvent was removed under reduced pressure and the residuesobtained were purified by silica gel column chromatography[chloroform-chloroform:methanol=9:1 (v/v)] to yield the titled compoundas a pale yellow solid (0.100 g, 38%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.83 (3H, t, J=7.2 Hz), 1.09-1.21 (2H, m),1.33-1.47 (4H, m), 1.75-1.90 (2H, m), 2.00-2.23 (4H, m), 2.36 (3H, s),2.69-2.81 (1H, m), 2.94-3.03 (1H, m), 3.06-3.22 (2H, m), 3.23-3.74 (8H,m), 3.98 (2H, s), 4.39-4.50 (1H, m), 4.60 (2H, d, J=6.7 Hz), 5.17 (2H,s), 5.39 (2H, s), 5.53-5.61 (1H, m), 6.50 (1H, s), 6.96 (2H, s),7.11-7.24 (5H, m), 7.28 (1H, s), 7.75 (1H, d, J=11.0 Hz), 7.97 (1H, t,J=5.7 Hz), 8.03 (1H, t, J=5.9 Hz), 8.09 (1H, d, J=7.8 Hz), 8.27 (1H, t,J=6.5 Hz), 8.48 (1H, d, J=9.0 Hz), 8.60 (1H, t, J=6.5 Hz).

MS (ESI) m/z: 1034 (M+H)⁺

Process 9: Antibody-Drug Conjugate (55)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.61mLmg⁻¹cm⁻¹ was used) described in Production method 1. The solution(1.25 mL) was placed in a 1.5 mL polypropylene tube and charged with anaqueous solution of 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.)(0.025 mL; 3.0 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (Nacalai Tesque, Inc.;0.0625 mL). After confirming that the solution had pH of 7.4±0.1, thedisulfide bond at hinge part in the antibody was reduced by incubatingat 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (Sigma-Aldrich Co. LLC; 0.109 mL) and a dimethyl sulfoxidesolution containing 10 mM of the compound obtained in above Process 8(0.039 mL; 4.6 equivalents per antibody molecule) to the above solutionat room temperature, it was stirred by using a tube rotator (MTR-103,manufactured by AS ONE Corporation) for conjugating the drug linker tothe antibody at room temperature for 40 minutes. Next, an aqueoussolution (0.008 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was addedthereto and stirred to terminate the raction of drug linker at roomtemperature for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate. After that, the solution was concentrated bythe Common procedure A described in Production method 1.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370) 19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 12.57 mg/mL, antibody yield: 8.8 mg (70%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.2.

Example 59 Antibody-Drug Conjugate (56)

Process 1: Antibody-Drug Conjugate (56)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.61mLmg⁻¹cm⁻¹ was used) described in Production method 1. The solution(1.25 mL) was placed in a 1.5 mL polypropylene tube and charged with anaqueous solution of 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.)(0.051 mL; 6.0 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (Nacalai Tesque, Inc.;0.0625 mL). After confirming that the solution had pH of 7.4±0.1, thedisulfide bond at hinge part in the antibody was reduced by incubatingat 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (Sigma-Aldrich Co. LLC; 0.067 mL) a dimethyl sulfoxidesolution containing and 10 mM of the compound obtained in Process 8 ofExample 58 (0.085 mL; 10.0 equivalents per antibody molecule) to theabove solution at room temperature, it was stirred by using a tuberotator (MTR-103, manufactured by AS ONE Corporation) for conjugatingthe drug linker to the antibody at room temperature for 60 minutes.Next, an aqueous solution (0.013 mL) of 100 mM NAC (Sigma-Aldrich Co.LLC) was added thereto and stirred to terminate the raction of druglinker at room temperature for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 1.33 mg/mL, antibody yield: 7.98 mg (64%), andaverage number of conjugated drug molecules (n) per antibody molecule:4.9.

Example 60 Antibody-Drug Conjugate (57)

Process 1: Antibody-Drug Conjugate (57)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 2 was prepared to have antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.61mLmg⁻¹cm⁻¹ was used) described in Production method 1. The solution(1.25 mL) was placed in a 1.5 mL polypropylene tube and charged with anaqueous solution of 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.)(0.051 mL; 6.0 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (Nacalai Tesque, Inc.;0.0625 mL). After confirming that the solution had pH of 7.4±0.1, thedisulfide bond at hinge part in the antibody was reduced by incubatingat 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding dimethylsulfoxide (Sigma-Aldrich Co. LLC; 0.025 mL) and a dimethyl sulfoxidesolution containing 10 mM of the compound obtained in Process 8 ofExample 58 (0.127 mL; 15.0 equivalents per antibody molecule) to theabove solution at room temperature, it was stirred by using a tuberotator (MTR-103, manufactured by AS ONE Corporation) for conjugatingthe drug linker to the antibody at room temperature for 60 minutes.Next, an aqueous solution (0.019 mL) of 100 mM NAC (Sigma-Aldrich Co.LLC) was added thereto and stirred to terminate the raction of druglinker at room temperature for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate. After that, the solution was concentrated bythe Common procedure A described in Production method 1.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5000 (measured average value), andε_(D,370)=19000 (measured average value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 0.91 mg/mL, antibody yield: 5.46 mg (44%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.3.

Example 61 Antibody-Drug Conjugate (58)

Almost the whole amounts of the antibody-drug conjugates of Examples 59and 60 were mixed and the solution was concentrated by the Commonprocedure A described in Production method 1 to yield the titledantibody-drug conjugate.

Antibody concentration: 10.0 mg/mL, antibody yield: 12.30 mg, andaverage number of conjugated drug molecules (n) per antibody molecule:5.4.

Example 62 Antibody-Drug Conjugate (59)

Process 1: Antibody-Drug Conjugate (59)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 1 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (100 mL, 1 g of the antibody) wasplaced in a 250 mL flask and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (2.43 mL; 3.6 equivalents perantibody molecule) and further with an aqueous solution of 1 Mdipotassium hydrogen phosphate (5 mL). After confirming that thesolution had pH near 7.4 by using a pH meter, the disulfide bond athinge part in the antibody was reduced by incubating at 37° C. for 1hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process8 of Example 58 (3.51 mL; 5.2 equivalents per antibody molecule) anddimethyl sulfoxide (2.14 mL) to the above solution at room temperature,it was stirred with a stirrer for conjugating the drug linker to theantibody in a water bath at 15° C. for 130 minutes. Next, an aqueoussolution (0.547 mL) of 100 mM NAC was added thereto and furtherincubated to terminate the raction of drug linker at room temperaturefor 20 minutes.

Purification: The above solution was subjected to ultrafiltrationpurification using an ultrafiltration apparatus composed of anultrafiltration membrane (Merck Japan, Pellicon XL Cassette, Biomax 50KDa), a tube pump (Cole-Parmer International, MasterFlex Pump model77521-40, Pump Head model 7518-00), and a tube (Cole-ParmerInternational, MasterFlex Tube L/S16). Specifically, while ABS was addeddropwise (a total of 800 mL) as a buffer solution for purification tothe reaction solution, ultrafiltration purification was performed forremoving unconjugated drug linkers and other low-molecular-weightreagents, also replacing the buffer solution with ABS, and furtherconcentrating the solution, to yield about 70 mL of a solutioncontaining the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5178 (measured value), and ε_(D,370)=20217(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 14.2 mg/mL, antibody yield: 1.0 g (about 100%),and average number of conjugated drug molecules (n) per antibodymolecule: 3.2.

Example 63 Antibody-Drug Conjugate (60)

Process 1: Antibody-Drug Conjugate (60)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 1 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (5 mL, 50 mg of the antibody) wasplaced in a 15 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.075 mL; 4 equivalents perantibody molecule). After confirming that the solution had pH near 7.0by using a pH meter, the disulfide bond at hinge part in the antibodywas reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process8 of Example 58 (0.219 mL; 6.5 equivalents per antibody molecule) anddimethyl sulfoxide (0.064 mL) to the above solution, it was incubatedfor conjugating the drug linker to the antibody in a water bath at 15°C. for 90 minutes. Next, an aqueous solution (0.033 mL; 9.8 equivalentsper antibody molecule) of 100 mM NAC was added thereto and incubated toterminate the raction of drug linker at room temperature for another 20minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 19 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5178 (measured value), and ε_(D,370)=20217(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 2.19 mg/mL, antibody yield: 42 mg (83%), andaverage number of conjugated drug molecules (n) per antibody molecule:4.7.

Example 64 Antibody-Drug Conjugate (61)

Process 1: Antibody-Drug Conjugate (61)

Reduction of the antibody: The M30-H1-L4P antibody produced in ReferenceExample 1 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (4 mL, 40 mg of the antibody) wasplaced in a 15 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.14 mL; 5.2 equivalents perantibody molecule). After confirming that the solution had pH near 7.0by using a pH meter, the disulfide bond at hinge part in the antibodywas reduced by incubating at 37° C. for 1 hour. Conjugation betweenantibody and drug linker: After adding a dimethyl sulfoxide solutioncontaining 10 mM of the compound obtained in Process 8 of Example 58(0.232 mL; 8.6 equivalents per antibody molecule) to the above solution,it was incubated for conjugating the drug linker to the antibody in awater bath at 15° C. for 60 minutes. Next, an aqueous solution (0.035mL; 12.9 equivalents per antibody molecule) of 100 mM NAC was addedthereto and incubated to terminate the raction of drug linker at roomtemperature for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 13 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5178 (measured value), and ε_(D,370)=20217(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 2.03 mg/mL, antibody yield: 26 mg (66%), andaverage number of conjugated drug molecules (n) per antibody molecule:5.7.

Example 65 Antibody-Drug Conjugate (62)

Process 1: Antibody-Drug Conjugate (62)

Reduction of the antibody: The M30-H1-L4 antibody produced in ReferenceExample 1 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (1.25 mL, 12.5 mg of the antibody)was placed in a 1.5 mL tube and charged with an aqueous solution of 10mM TCEP (Tokyo Chemical Industry Co., Ltd.) (0.0287 mL; 3.4 equivalentsper antibody molecule) and an aqueous solution of 1 M dipotassiumhydrogen phosphate (Nacalai Tesque, Inc.; 0.0625 mL). After confirmingthat the solution had pH of 7.4±0.1, the disulfide bond at hinge part inthe antibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process8 of Example 58 (0.0439 mL; 5.2 equivalents per antibody molecule) anddimethyl sulfoxide (0.0267 mL) to the above solution at roomtemperature, it was incubated for conjugating the drug linker to theantibody in a water bath at 15° C. for 1 hour. Next, an aqueous solution(0.0066 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was added thereto andincubated to terminate the raction of drug linker at room temperaturefor another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate. After that, the solution was concentrated bythe Common procedure A.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5178 (measured value), and ε_(D,370)=20217(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 10.0 mg/mL, antibody yield: 7.8 mg (62%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.4.

Example 66 Antibody-Drug Conjugate (63)

Process 1: Antibody-Drug Conjugate (63)

Reduction of the antibody: The M30-H1-L4 antibody produced in ReferenceExample 1 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.61 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (1.25 mL, 12.5 mg of the antibody)was placed in a 1.5 mL tube and charged with an aqueous solution of 10mM TCEP (Tokyo Chemical Industry Co., Ltd.) (0.0439 mL; 5.2 equivalentsper antibody molecule) (0.0287 mL; 3.4 equivalents per antibodymolecule) and an aqueous solution of 1 M dipotassium hydrogen phosphate(Nacalai Tesque, Inc.; 0.0625 mL). After confirming that the solutionhad pH of 7.4±0.1, the disulfide bond at hinge part in the antibody wasreduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process8 of Example 58 (0.0726 mL; 8.6 equivalents per antibody molecule) tothe above solution at room temperature, it was incubated for conjugatingthe drug linker to the antibody in a water bath at 15° C. for 1 hour.Next, an aqueous solution (0.011 mL) of 100 mM NAC (Sigma-Aldrich Co.LLC) was added thereto and incubated to terminate the raction of druglinker at room temperature for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method to yield 6 mL of a solution containing the titledantibody-drug conjugate. After that, the solution was concentrated bythe Common procedure A.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=235300 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5178 (measured value), and ε_(D,370)=20217(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 10.0 mg/mL, antibody yield: 7.3 mg (58%), andaverage number of conjugated drug molecules (n) per antibody molecule:5.4.

Example 67 Antibody-Drug Conjugate (64)

Process 1: Antibody-Drug Conjugate (64)

Reduction of the antibody: The anti-CD30 antibody produced in ReferenceExample 3 was prepared to have antibody concentration of 10 mg/mL withPBS6.5/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.75 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (0.4 mL, 4 mg of the antibody) wasplaced in a 1.5 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.0065 mL; 2.5 equivalents perantibody molecule) and an aqueous solution of 1 M dipotassium hydrogenphosphate (Nacalai Tesque, Inc.; 0.0058 mL). After confirming that thesolution had pH of 7.0±0.1, the disulfide bond at hinge part in theantibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process8 of Example 58 (0.0116 mL; 4.5 equivalents per antibody molecule) anddimethyl sulfoxide (0.0101 mL) to the above solution at roomtemperature, it was stirred by using a tube rotator (MTR-103,manufactured by AS ONE Corporation) for conjugating the drug linker tothe antibody at room temperature for 1 hour. Next, an aqueous solution(0.0017 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was added thereto andfurther incubated to terminate the raction of drug linker at roomtemperature for 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method 1 to yield 2.5 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=270400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5178 (measured value), and ε_(D,370)=20217(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 0.96 mg/mL, antibody yield: 2.4 mg (60%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.7.

Example 68 Antibody-Drug Conjugate (65)

Process 1: Antibody-Drug Conjugate (65)

Reduction of the antibody: The anti-CD30 antibody produced in ReferenceExample 3 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.75 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (0.4 mL, 4 mg of the antibody) wasplaced in a 1.5 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.0129 mL; equivalents perantibody molecule) and an aqueous solution of 1 M dipotassium hydrogenphosphate (Nacalai Tesque, Inc.; 0.006 mL). After confirming that thesolution had pH of 7.0±0.1, the disulfide bond at hinge part in theantibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process8 of Example 58 (0.0233 mL; 9 equivalents per antibody molecule) to theabove solution at room temperature, it was stirred by using a tuberotator (MTR-103, manufactured by AS ONE Corporation) for conjugatingthe drug linker to the antibody at room temperature for 1 hour. Next, anaqueous solution (0.0035 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) wasadded thereto and further incubated to terminate the raction of druglinker at room temperature for 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method 1 to yield 2.5 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=270400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5178 (measured value), and ε_(D,370)=20217(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 0.39 mg/mL, antibody yield: 1.0 mg (24%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.8.

Example 69 Antibody-Drug Conjugate (66)

Process 1: Antibody-Drug Conjugate (66)

Reduction of the antibody: The anti-CD33 antibody produced in ReferenceExample 4 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.66 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (0.4 mL, 4 mg of the antibody) wasplaced in a 1.5 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.0065 mL; 2.5 equivalents perantibody molecule) and an aqueous solution of 1 M dipotassium hydrogenphosphate (Nacalai Tesque, Inc.; 0.0058 mL). After confirming that thesolution had pH of 7.0±0.1, the disulfide bond at hinge part in theantibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process8 of Example 58 (0.0116 mL; 4.5 equivalents per antibody molecule) anddimethyl sulfoxide (0.0101 mL) to the above solution at roomtemperature, it was stirred by using a tube rotator (MTR-103,manufactured by AS ONE Corporation) for conjugating the drug linker tothe antibody at room temperature for 1 hour. Next, an aqueous solution(0.0017 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was added thereto andfurther incubated to terminate the raction of drug linker at roomtemperature for 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method 1 to yield 2.5 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=256400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5178 (measured value), and ε_(D,370)=20217(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 1.19 mg/mL, antibody yield: 3.0 mg (74%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.8.

Example 70 Antibody-Drug Conjugate (67)

Process 1: Antibody-Drug Conjugate (67)

Reduction of the antibody: The anti-CD33 antibody produced in ReferenceExample 4 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.66 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (0.4 mL, 4 mg of the antibody) wasplaced in a 1.5 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.0129 mL; equivalents perantibody molecule) and an aqueous solution of 1 M dipotassium hydrogenphosphate (Nacalai Tesque, Inc.; 0.006 mL). After confirming that thesolution had pH of 7.0±0.1, the disulfide bond at hinge part in theantibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process8 of Example 58 (0.0233 mL; 9 equivalents per antibody molecule) to theabove solution at room temperature, it was stirred by using a tuberotator (MTR-103, manufactured by AS ONE Corporation) for conjugatingthe drug linker to the antibody at room temperature for 1 hour. Next, anaqueous solution (0.0035 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) wasadded thereto and further incubated to terminate the raction of druglinker at room temperature for 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method 1 to yield 2.5 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=256400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5178 (measured value), and ε_(D,370)=20217(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 1.24 mg/mL, antibody yield: 3.1 mg (78%), andaverage number of conjugated drug molecules (n) per antibody molecule:7.0.

Example 71 Antibody-Drug Conjugate (68)

Process 1: Antibody-Drug Conjugate (68)

Reduction of the antibody: The anti-CD70 antibody produced in ReferenceExample 5 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.69 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (0.4 mL, 4 mg of the antibody) wasplaced in a 1.5 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.0065 mL; 2.5 equivalents perantibody molecule) and an aqueous solution of 1 M dipotassium hydrogenphosphate (Nacalai Tesque, Inc.; 0.0058 mL). After confirming that thesolution had pH of 7.0±0.1, the disulfide bond at hinge part in theantibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process8 of Example 58 (0.0116 mL; 4.5 equivalents per antibody molecule) anddimethyl sulfoxide (0.0101 mL) to the above solution at roomtemperature, it was stirred by using a tube rotator (MTR-103,manufactured by AS ONE Corporation) for conjugating the drug linker tothe antibody at room temperature for 1 hour. Next, an aqueous solution(0.0017 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) was added thereto andfurther incubated to terminate the raction of drug linker at roomtemperature for 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method 1 to yield 2.5 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,28)=262400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5178 (measured value), and ε_(D,370)=20217(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 1.10 mg/mL, antibody yield: 2.8 mg (69%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.8.

Example 72 Antibody-Drug Conjugate (69)

Process 1: Antibody-Drug Conjugate (69)

Reduction of the antibody: The anti-CD70 antibody produced in ReferenceExample 5 was prepared to have antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure B (as absorption coefficientat 280 nm, 1.69 mLmg⁻¹cm⁻¹ was used) and Common procedure C-1 describedin Production method 1. The solution (0.4 mL, 4 mg of the antibody) wasplaced in a 1.5 mL tube and charged with an aqueous solution of 10 mMTCEP (Tokyo Chemical Industry Co., Ltd.) (0.0129 mL; equivalents perantibody molecule) and an aqueous solution of 1 M dipotassium hydrogenphosphate (Nacalai Tesque, Inc.; 0.006 mL). After confirming that thesolution had pH of 7.0±0.1, the disulfide bond at hinge part in theantibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a dimethylsulfoxide solution containing 10 mM of the compound obtained in Process8 of Example 58 (0.0233 mL; 9 equivalents per antibody molecule) to theabove solution at room temperature, it was stirred by using a tuberotator (MTR-103, manufactured by AS ONE Corporation) for conjugatingthe drug linker to the antibody at room temperature for 1 hour. Next, anaqueous solution (0.0035 mL) of 100 mM NAC (Sigma-Aldrich Co. LLC) wasadded thereto and further incubated to terminate the raction of druglinker at room temperature for 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as buffer solution) described inProduction method 1 to yield 2.5 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1 (as molar absorption coefficient,ε_(A,280)=262400 (estimated calculation value), ε_(A,370)=0 (estimatedcalculation value), ε_(D,280)=5178 (measured value), and ε_(D,370)=20217(measured value) were used), the following characteristic values wereobtained.

Antibody concentration: 1.16 mg/mL, antibody yield: 2.9 mg (73%), andaverage number of conjugated drug molecules (n) per antibody molecule:7.0.

Example 73 (Another Method for Synthesizing Compound of Process 8 ofExample 58)

Process 1: tert-ButylN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalaninate

Under ice cooling, to THF (12.0 ml) solution of tert-butylN-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenyl alaninate (J.Pept. Res., 1999, vol. 53, pp. 393) (0.400 g, 0.717 mmol),1,8-diazabicyclo[5.4.0]-7-undecene (0.400 ml) was added and stirred atroom temperature for 4 days, and then N-succinimidyl 6-maleimidehexanoate (0.221 g, 0.717 mmol) was further added and stirred for 3hours. The reaction solution was diluted with ethyl acetate and washedwith an aqueous solution of 10% citric acid, a saturated aqueoussolution of sodium hydrogen carbonate, and saturated brine, and then theorganic layer was dried over anhydrous magnesium sulfate. After thesolvent was removed under reduced pressure, the residues obtained werepurified by silica gel column chromatography[chloroform-chloroform:methanol=9:1 (v/v)] to yield the titled compoundas a pale yellow solid (0.295 g, 78%).

¹H-NMR (400 MHz, CDCl₃) δ: 1.28-1.36 (2H, m), 1.41 (9H, s), 1.57-1.71(4H, m), 2.23 (2H, t, J=7.6 Hz), 3.09 (2H, d, J=6.0 Hz), 3.51 (2H, t,J=7.6 Hz), 3.85-4.02 (4H, m), 4.69-4.78 (1H, m), 6.15 (1H, t, J=4.6 Hz),6.33 (1H, d, J=7.3 Hz), 6.60 (1H, t, J=5.0 Hz), 6.68 (2H, s), 7.10-7.16(2H, m), 7.22-7.31 (3H, m).

MS (ESI) m/z: 529 (M+H)⁺

Process 2:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanine

To a dichloromethane (8.00 ml) solution of the compound (0.295 g, 0.558mmol) obtained in Process 1 above, trifluoroacetic acid (4.00 mL) wasadded and stirred at room temperature for 18 hours. The solvent wasremoved under reduced pressure to yield the titled compound as a paleyellow solid (0.240 g, 91%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.15-1.23 (2H, m), 1.40-1.53 (4H, m), 2.10(2H, t, J=7.6 Hz), 2.88 (1H, dd, J=13.7, 8.9 Hz), 3.04 (1H, dd, J=13.7,5.0 Hz), 3.35-3.43 (2H, m), 3.58-3.77 (4H, m), 4.41 (1H, td, J=7.8, 5.0Hz), 7.00 (2H, s), 7.16-7.31 (5H, m), 8.00 (1H, t, J=5.7 Hz), 8.06 (1H,t, J=5.7 Hz), 8.13 (1H, d, J=7.8 Hz).

Process 3:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

The compound (0.572 g, 1.21 mmol) obtained in Process 2 above wasdissolved in dichloromethane (12.0 mL), charged withN-hydroxysuccinimide (0.152 g, 1.32 mmol) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.253 g,1.32 mmol), and stirred for 1 hour. The reaction solution was added toan N,N-dimethylformamide (22.0 mL) solution of the mixture (1.10 mmol)obtained in Process 5 of Example 58, and stirred at room temperature for3 hours. The reaction solution was charged with an aqueous solution of10% citric acid and extracted with chloroform. The obtained organiclayer was dried over sodium sulfate and filtered. The solvent wasremoved under reduced pressure and the residues obtained were purifiedby silica gel column chromatography [chloroform chloroform:methanol=8:2(v/v)] to yield the titled compound as a pale yellow solid (0.351 g,31%). The instrumental data of the compound was the same as that of thecompound of Process 8 of Example 58.

Example 74 (Another Method for Synthesizing Compound of Process 8 ofExample 58)

Process 1: Benzyl [({N-[(9H-fluoren-9-ylmethoxy) carbonyl]glycyl}amino)methoxy]acetate

To a tetrahydrofuran (200 ml) solution of the compound (7.37 g, 20.0mmol) obtained in Process 1 of Example 58, benzyl glycolate (6.65 g,40.0 mmol) and p-toluene sulfonic acid monohydrate (0.381 g, 2.00 mmol)were added at 0° C. and stirred at room temperature for 2 hours and 30minutes. The reaction solution was charged with a saturated aqueoussolution of sodium hydrogen carbonate and extracted with ethyl acetate.The obtained organic layer was dried over sodium sulfate and filtered.The solvent was removed under reduced pressure and the residues obtainedwere purified by silica gel column chromatography [hexane:ethylacetate=100:0 (v/v)-0:100] to yield the titled compound as a colorlesssolid (6.75 g, 71%). The instrumental data of the compound was the sameas that of the compound of Process 2 of Example 58.

Process 2:N-[(benzyloxy)carbonyl]glycylglycyl-L-phenylalanine-N-{[(2-(benzyloxy)-2-oxoethoxy]methyl}glycinamide

To an N,N-dimethylformamide (140 mL) solution of the compound (6.60 g,13.9 mmol) obtained in Process 1 above,1,8-diazabicyclo[5.4.0]undec-7-ene (2.22 g, 14.6 mmol) was added at 0°C. and stirred at room temperature for 15 minutes. The reaction solutionwas charged with an N,N-dimethylformamide (140 mL) solution ofN-[(benzyloxy)carbonyl]glycylglycyl-L-phenylalanine (6.33 g, 15.3 mmol),N-hydroxysuccinimide (1.92 g, 16.7 mmol), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.20 g,16.7 mmol) stirred in advance at room temperature for 1 hour, andstirred at room temperature for 4 hours. The reaction solution wascharged with 0.1 N hydrochloric acid and extracted with chloroform. Theobtained organic layer was dried over sodium sulfate and filtered. Thesolvent was removed under reduced pressure and the residues obtainedwere purified by silica gel column chromatography[chloroform-chloroform:methanol=8:2 (v/v)] to yield the titled compoundas a colorless solid (7.10 g, 79%).

¹H-NMR (DMSO-D₆) δ: 2.78 (1H, dd, J=13.9, 9.6 Hz), 3.05 (1H, dd, J=13.9,4.5 Hz), 3.56-3.80 (6H, m), 4.15 (2H, s), 4.47-4.55 (1H, m), 4.63 (2H,d, J=6.6 Hz), 5.03 (2H, s), 5.15 (2H, s), 7.16-7.38 (15H, m), 7.52 (1H,t, J=5.9 Hz), 8.03 (1H, t, J=5.5 Hz), 8.17 (1H, d, J=8.2 Hz), 8.36 (1H,t, J=5.7 Hz), 8.61 (1H, t, J=6.6 Hz).

Process 3:Glycylglycyl-L-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide

To an N,N-dimethylformamide (216 mL) solution of the compound (7.00 g,10.8 mmol) obtained in Process 2 above, palladium carbon catalyst (7.00g) was added and stirred under hydrogen atmosphere at room temperaturefor 24 hours. The insolubles were removed by filtration through Celite,and the solvent was removed under reduced pressure. The residuesobtained were dissolved in water, the insoluble material was removed byfiltration through Celite, and the solvent was removed under reducedpressure. This procedure was repeated twice to yield the titled compoundas a colorless solid (3.77 g, 82%).

¹H-NMR (DMSO-D₅) δ: 2.84 (1H, dd, J=13.7, 9.8 Hz), 3.08 (1H, dd, J=13.7,4.7 Hz), 3.50-3.72 (4H, m), 3.77-3.86 (2H, m), 3.87 (2H, s), 4.52-4.43(1H, m), 4.61 (2H, d, J=6.6 Hz), 7.12-7.30 (5H, m), 8.43 (1H, t, J=5.9Hz), 8.54 (1H, d, J=7.8 Hz), 8.70 (1H, t, J=6.3 Hz), 8.79 (1H, t, J=5.5Hz).

Process 4:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide

To an N,N-dimethylformamide (85.0 mL) solution of the compound (3.59 g,8.48 mmol) obtained in Process 3 above, N-succinimidyl 6-maleimidehexanoate (2.88 g, 9.33 mmol) and triethylamine (0.858 g, 8.48 mmol)were added and stirred at room temperature for 1 hour. The reactionsolution was charged with 0.1 N hydrochloric acid and extracted withchloroform and a mixed solvent of chloroform and methanol[chloroform:methanol=4:(v/v)]. The obtained organic layer was dried oversodium sulfate and filtered. The solvent was removed under reducedpressure and the residues obtained were purified by silica gel columnchromatography [chloroform-partitioned organic layer ofchloroform:methanol:water=7:3:1 (v/v/v)] to yield the titled compound asa colorless solid (3.70 g, 71%).

¹H-NMR (DMSO-D₆) δ: 1.13-1.24 (2H, m), 1.42-1.53 (4H, m), 2.11 (2H, t,J=7.4 Hz), 2.80 (1H, dd, J=13.7, 9.8 Hz), 3.06 (1H, dd, J=13.9, 4.5 Hz),3.37 (2H, t, J=7.2 Hz), 3.56-3.78 (6H, m), 3.97 (2H, s), 4.46-4.53 (1H,m), 4.61 (2H, d, J=6.3 Hz), 7.00 (2H, s), 7.15-7.29 (5H, m), 8.03-8.20(3H, m), 8.32 (1H, t, J=5.9 Hz), 8.60 (1H, t, J=6.7 Hz).

Process 5:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

To an N,N-dimethylformamide (40.0 mL) solution of mesylate of thecompound (4) (1.14 g, 2.00 mmol), triethylamine (0.202 g, 2.00 mmol),the compound (1.48 g, 2.40 mmol) obtained in Process 4 above, and4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(0.993 g, 3.00 mmol) containing 16.4% water were added at 0° C. andstirred at room temperature for 1 hour. The solvent was removed underreduced pressure and the residues obtained were purified by silica gelcolumn chromatography [chloroform-chloroform:methanol=8:2 (v/v)] toyield the titled compound as a pale yellow solid (1.69 g, 82%). Theinstrumental data of the compound was the same as that of the compoundof Process 8 of Example 58.

Example 75N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-2-hydroxyacetamide

Process 1:2-{[(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethylacetate

Under ice cooling, to an N,N-dimethylformamide (20.0 mL) suspension ofmesylate of the compound (4) (0.500 g, 0.941 mmol),N,N-diisopropylethylamine (0.492 mL, 2.82 mmol) and acetoxyacetylchloride (0.121 ml, 1.13 mmol) were added and stirred at roomtemperature for 1 hour. The solvent was removed under reduced pressureand the residues obtained were purified by silica gel columnchromatography [chloroform partitioned organic layer ofchloroform:methanol:water=7:3:1 (v/v/v)] to yield the titled compound asa pale yellow solid (0.505 g, quantitative).

¹H-NMR (400 MHz, DMSO-d₅) δ: 0.87 (3H, t, J=7.4 Hz), 1.81-1.92 (2H, m),2.08 (3H, s), 2.08-2.22 (2H, m), 2.41 (3H, s), 3.14-3.21 (2H, m), 4.51(2H, dd, J=19.4, 14.7 Hz), 5.22 (2H, dd, J=40.1, 19.0 Hz), 5.43 (2H, s),5.56-5.61 (1H, m), 6.53 (1H, s), 7.31 (1H, s), 7.81 (1H, d, J=11.0 Hz),8.67 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 536 (M+H)⁺

Process 2:N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-2-hydroxyacetamide

To a methanol (50.0 mL) suspension of the compound (0.504 g, 0.941 mmol)obtained in Process 1 above, tetrahydrofuran (20.0 ml) and an aqueoussolution of 1 N sodium hydroxide (4.00 ml, 4.00 mmol) were added andstirred at room temperature for 1 hour. The reaction was terminated bythe addition of 1 N hydrochloric acid (5.00 ml, 5.00 mmol), and thesolvent was removed under reduced pressure. The residues obtained werepurified by silica gel column chromatography [chloroform partitionedorganic layer of chloroform:methanol:water=7:3:1 (v/v/v)] to yield thetitled compound as a pale yellow solid (0.412 g, 89%). This compound wasconfirmed in the tumor of a cancer-bearing mouse that received theantibody-drug conjugate (55) or (56).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.3 Hz), 1.78-1.95 (2H, m),2.09-2.28 (2H, m), 2.39 (3H, s), 3.07-3.27 (2H, m), 3.96 (2H, d, J=6.0Hz), 5.11-5.26 (2H, m), 5.42 (2H, s), 5.46-5.54 (1H, m), 5.55-5.63 (1H,m), 6.52 (1H, s), 7.30 (1H, s), 7.78 (1H, d, J=10.9 Hz), 8.41 (1H, d,J=9.1 Hz). MS (ESI) m/z: 494 (M+H)⁺

Example 76 (Another Method for Synthesizing Compound of Example 75)

Process 1:N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-2-hydroxyacetamide

Glycolic acid (0.0201 g, 0.27 mmol) was dissolved inN,N-dimethylformamide (1.0 mL), charged with N-hydroxysuccinimide(0.0302 g, 0.27 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (0.0508 g, 0.27 mmol), and stirred for 1 hour. Thereaction solution was added to an N,N-dimethylformamide suspension (1.0mL) charged with mesylate of the compound (4) (0.1 g, 0.176 mmol) andtriethylamine (0.025 mL, 0.18 mmol) and stirred at room temperature for24 hours. The solvent was removed under reduced pressure and theresidues obtained were purified by silica gel column chromatography[chloroform chloroform:methanol=10:1 (v/v)] to yield the titled compoundas a pale yellow solid (0.080 g, 92%). The instrumental data of thecompound was the same as that of the compound obtained in Process 2 ofExample 75.

(Test Example 1) Production of Full-Length Human B7-H3 variant 1expression vector

cDNA encoding human B7-H3 variant 1 was amplified by PCR reaction usingcDNA synthesized from LNCaP cell (American Type Culture Collection:ATCC) total RNA as a template and the following primer set:

primer 1: (SEQ ID NO: 22)5′-ctatagggagacccaagctggctagcatgctgcgtcggcggggca g-3′ and primer 2:(SEQ ID NO: 23) 5′-aacgggccctctagactcgagcggccgctcaggctatttcttgtccatcatcttctttgctgtcag-3′.

Next, the obtained PCR product was purified by using MagExtractor PCR &Gel cleanup (Toyobo Co., Ltd.). The purified product was furtherdigested with restriction enzymes (NheI/NotI) and thereafter purified byusing MagExtractor PCR & Gel cleanup (Toyobo Co., Ltd.). pcDNA3.1 (+)plasmid DNA (Life Technologies) was digested with the same restrictionenzymes as above (NheI/NotI) and thereafter purified by usingMagExtractor PCR & Gel cleanup (Toyobo Co., Ltd.).

These purified DNA solutions were mixed, further charged with Ligationhigh (Toyobo Co., Ltd.), and incubated for ligation at 16° C. for 8hours.

Escherichia coli DH5a competent cells (Life Technologies) weretransformed by the addition of the obtained reaction product.

The colonies thus obtained were subjected to colony direct PCR using PCRprimers and BGH reverse primer to select candidate clones.

The obtained candidate clones were cultured in a liquid medium (LB/Amp),and plasmid DNA was extracted with MagExtractor-Plasmid-(Toyobo Co.,Ltd.).

Each obtained clone was compared with the provided CDS sequence by thesequencing analysis between primer 3 (CMV promoter primer):

(SEQ ID NO: 24) 5′-cgcaaatgggcggtaggcgtg-3′ andprimer 4 (BGH reverse primer): (SEQ ID NO: 25) 5′-tagaaggcacagtcgagg-3′with the obtained plasmid DNA as a template.

After confirming the sequence, the obtained clone was cultured in 200 mLof LB/Amp medium, and plasmid DNA was extracted by using VioGene PlasmidMidi V-100 kit.

The vector was designated as pcDNA3.1-B7-H3. The sequence of an ORF siteof the B7-H3 variant 1 gene cloned in the vector is shown in nucleotidepositions 1 to 1602 of SEQ ID NO: 26 (FIG. 16) in the Sequence Listing.Also, the amino acid sequence of the B7-H3 variant 1 is shown in SEQ IDNO: 1 in the Sequence Listing.

(Test Example 2) Preparation of CCRF-CEM Cell Stably Expressing B7-H3Variant 1 Gene

pcDNA3.1-B7-H3 produced in Test Example 1 was transfected into CCRF-CEMcells (ATCC) by electroporation using Nucleofector II (manufactured byLonza Group Ltd.). Then, the cells were further cultured for two nightsin RPMI1640 medium (Life Technologies) containing 10% fetal bovine serum(FBS) (hereinafter, referred to as 10% FBS-RPMI1640) under conditions of37° C. and 5% CO₂.

After the 2-day culture, culture was started in 10% FBS-RPMI1640containing 750 μg/mL G418 (Life Technologies) in order to selectCCRF-CEM cells in which pcDNA3.1-B7-H3 was stably integrated.

After the 1-month culture, cloning was carried out by the limitingdilution method in order to yield a single cell clone. Specifically,cells having resistance to G418 were diluted into 10 cells/mL,inoculated to a 96-well plate at a concentration of 100 μL/well, andcultured, and cells allowed to proliferate were recovered fromindividual wells.

Flow cytometry was used for confirming B7-H3 expression in eachrecovered clone. Specifically, each recovered clone was washed twicewith PBS containing 5% FBS, thereafter suspended by the addition of PBScontaining 5% FBS and 10 μg/mL M30, and left standing at 4° C. for 30minutes. The clone was washed twice with PBS containing 5% FBS,thereafter suspended by the addition of Fluorescein-conjugated goat IgGfraction to mouse IgG (Whole Molecule) (#55493, manufactured by ICNPharmaceuticals, Inc.) diluted 1000-fold with PBS containing 5% FBS, andleft standing at 4° C. for 30 minutes. The clone was washed twice withPBS containing 5% FBS, thereafter resuspended in PBS containing 5% FBS,and detected by using a flow cytometer (FC500: Beckman Coulter, Inc.).

The CCRF-CEM cells stably expressing the B7-H3 variant 1 gene thusobtained by these procedures were designated as CEM_V1_3.1_2 cells. Theparent line CCRF-CEM cells were used as a cell line lacking B7-H3expression.

(Test Example 3) Cytotoxicity Test (1) of Antibody-Drug Conjugate

The CEM_V1_3.1_2 cells produced in Test Example 2 or CCRF-CEM cells(ATCC) were cultured in RPMI1640 (GIBCO) containing 10% fetal bovineserum (MOREGATE) (hereinafter, referred to as a medium). TheCEM_V1_3.1_2 cells or CCRF-CEM cells were prepared to have aconcentration of 8×10⁴ cells/mL by using a medium, added at aconcentration of 25 μL/well to a 96-well microplate for cell culturecharged with 65 μL/well of a medium, and cultured overnight. On the nextday, the M30-H1-L4 antibody, M30-H1-L4P antibody, and antibody-drugconjugate each diluted into 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32nM, and 0.064 nM by using a medium were added at a concentration of 10μL/well to the microplate. A medium was added at a concentration of 10μL/well to test substance non-supplemented wells. The cells werecultured under 5% CO₂ at 37° C. for 3 days. After the culture, themicroplate was taken out from the incubator and left standing at roomtemperature for 30 minutes. The culture solution was charged with anequal amount of CellTiter-Glo Luminescent Cell Viability Assay (Promega)and stirred. After the microplate was left standing at room temperaturefor 10 minutes, the amount of light emission was measured by using aplate reader (PerkinElmer). The IC₅₀ value was calculated according tothe following equation:

IC₅₀ (nM)=antilog((50−d)×(LOG₁₀ b−LOG₁₀ a)/(d−c)+LOG₁₀ b)

a: Concentration a of the test substance

b: Concentration b of the test substance

c: Ratio of live cells supplemented with the test substance having theconcentration a

d: Ratio of live cells supplemented with the test substance having theconcentration b

The concentrations a and b establish the relation a>b crossing 50% ratioof live cells.

The survival rate of cells at each concentration was calculatedaccording to the following equation:

Survival rate of cells (%)=a/b×100

a: Average amount of light emission from the test substance-supplementedwells (n=2)

b: Average amount of light emission from the test substancenon-supplemented wells (n=10)

The antibody-drug conjugates (5), (16), (21), (32), (44), (45), (46),(52), and (54) exhibited a cytotoxic activity of IC₅₀<0.1 (nM) againstthe CEM_V1_3.1_2 cells. The antibody-drug conjugates (1), (12), (13),(20), (28), (29), (35), (36), (37), (41), (49), and (53) exhibited acytotoxic activity of 0.1<IC₅₀<1 (nM) against the cells. Theantibody-drug conjugates (33), (34), (47), (48), (50), and (51)exhibited a cytotoxic activity of 1<IC₅₀<100 (nM) against the cells. Onthe other hand, none of these antibody-drug conjugates exhibited acytotoxic activity against the CCRF-CEM cells (>100 (nM)). Neither ofthe M30-H1-L4 antibody nor the M30-H1-L4P antibody exhibited a cytotoxicactivity against both of the cells (>100 (nM)).

(Test Example 4) Cytotoxicity Test (2) of Antibody-Drug Conjugate

Antigen-positive cells SR cells (ATCC) or antigen-negative cells Daudicells (ATCC) were cultured in RPMI1640 (GIBCO) containing 10% fetalbovine serum (MOREGATE) (hereinafter, referred to as a medium). The SRcells or Daudi cells were prepared to have a concentration of 2.8×10⁴cells/mL by using a medium and added at a concentration of 90 μL/well toa 96-well microplate for cell culture. Two hours later, the anti-CD30antibody and antibody-drug conjugates (6) and (7) each diluted into 40nM, 8 nM, 1.6 nM, 320 pM, 64 pM, 12.8 pM, and 2.6 pM by using a mediumwere added at a concentration of 10 μL/well to the microplate. A mediumwas added at a concentration of 10 μL/well to test substancenon-supplemented wells. The cells were cultured under 5% CO₂ at 37° C.for 3 days. After the culture, the microplate was taken out from theincubator and left standing at room temperature for 30 minutes. Theculture solution was charged with an equal amount of CellTiter-GloLuminescent Cell Viability Assay (Promega) and stirred. After themicroplate was left standing at room temperature for 10 minutes, theamount of light emission was measured by using a plate reader(PerkinElmer). The IC₅₀ value was calculated according to the followingequation: IC₅₀ (nM)=antilog((50−d)×(LOG₁₀b−LOG₁₀a)/(d−c)+LOG₁₀b)

a: Concentration a of the test substance

b: Concentration b of the test substance

c: Ratio of live cells supplemented with the test substance having theconcentration a

d: Ratio of live cells supplemented with the test substance having theconcentration b

The concentrations a and b establish the relation a>b crossing 50% ratioof live cells.

The survival rate of cells at each concentration was calculatedaccording to the following equation:

Survival rate of cells (%)=a/b×100

a: Average amount of light emission from the test substance-supplementedwells (n=2)

b: Average amount of light emission from the test substancenon-supplemented wells (n=12)

The antibody-drug conjugates (6) and (7) exhibited a cytotoxic activityof IC₅₀<0.01 (nM) against the SR cells. On the other hand, theantibody-drug conjugates (6) and (7) exhibited no cytotoxic activityagainst the Daudi cells (>4.0 (nM)). The anti-CD30 antibody exhibited nocytotoxic activity against both of the cells (>4.0 (nM)).

(Test Example 5) Cytotoxicity Test (3) of Antibody-Drug Conjugate

Antigen-positive cells SR cells (ATCC) were cultured in RPMI1640 (GIBCO)containing 10% fetal bovine serum (MOREGATE) (hereinafter, referred toas a medium). The SR cells were prepared to have a concentration of2.8×10⁴ cells/mL by using a medium and added at a concentration of 90μL/well to a 96-well microplate for cell culture. Two hours later, theanti-CD30 antibody and antibody-drug conjugates (22), (23), (38), (64),and (65) each diluted into 1000 nM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM,and 1 pM by using a medium were added at a concentration of 10 μL/wellto the microplate. A medium was added at a concentration of 10 μL/wellto test substance non-supplemented wells. The cells were cultured under5% CO₂ at 37° C. for 6 days. After the culture, the microplate was takenout from the incubator and left standing at room temperature for 30minutes. The culture solution was charged with an equal amount ofCellTiter-Glo Luminescent Cell Viability Assay (Promega) and stirred.After the microplate was left standing at room temperature for 10minutes, the amount of light emission was measured by using a platereader (PerkinElmer). The IC₅₀ value was calculated according to thefollowing equation:

IC₅₀ (nM)=antilog((50−d)×(LOG₁₀ b−LOG₁₀ a)/(d−c)+LOG₁₀ b)

a: Concentration a of the test substance

b: Concentration b of the test substance

c: Ratio of live cells supplemented with the test substance having theconcentration a

d: Ratio of live cells supplemented with the test substance having theconcentration b

The concentrations a and b establish the relation a>b crossing 50% ratioof live cells.

The survival rate of cells at each concentration was calculatedaccording to the following equation:

Survival rate of cells (%)=a/b×100

a: Average amount of light emission from the test substance-supplementedwells (n=2)

b: Average amount of light emission from the test substancenon-supplemented wells (n=12)

The antibody-drug conjugates (23), (38), (64), and (65) exhibited acytotoxic activity of IC₅₀<0.01 (nM) against the SR cells. Theantibody-drug conjugate (22) exhibited a cytotoxic activity of IC₅₀<0.1(nM) against the SR cells. The anti-CD30 antibody exhibited no cytotoxicactivity against the SR cells (>4.0 (nM)).

(Test Example 6) Cytotoxicity Test (4) of Antibody-Drug Conjugate

Antigen-positive cells HL-60 cells (ATCC) or antigen-negative cells Rajicells (ATCC) were cultured in RPMI1640 (GIBCO) containing 10% fetalbovine serum (MOREGATE) (hereinafter, referred to as a medium). TheHL-60 cells or Raji cells were prepared to have a concentration of 8×10⁴cells/mL by using a medium and added at a concentration of 25 μL/well toa 96-well microplate for cell culture charged with 65 μL/well of amedium. The anti-CD33 antibody and antibody-drug conjugates (8) and (9)each diluted into 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, and0.064 nM by using a medium were added at a concentration of 10 μL/wellto the microplate. A medium was added at a concentration of 10 μL/wellto test substance non-supplemented wells. The cells were cultured under5% CO₂ at 37° C. for 3 days. After the culture, the microplate was takenout from the incubator and left standing at room temperature for 30minutes. The culture solution was charged with an equal amount ofCellTiter-Glo Luminescent Cell Viability Assay (Promega) and stirred.After the microplate was left standing at room temperature for 10minutes, the amount of light emission was measured by using a platereader (PerkinElmer). The IC₅₀ value was calculated according to thefollowing equation:

IC₅₀ (nM)=antilog((50−d)×(LOG₁₀ b−LOG₁₀ a)/(d−c)+LOG₁₀ b)

a: Concentration a of the test substance

b: Concentration b of the test substance

c: Ratio of live cells supplemented with the test substance having theconcentration a

d: Ratio of live cells supplemented with the test substance having theconcentration b

The concentrations a and b establish the relation a>b crossing 50% ratioof live cells.

The survival rate of cells at each concentration was calculatedaccording to the following equation:

Survival rate of cells (%)=a/b×100

a: Average amount of light emission from the test substance-supplementedwells (n=2)

b: Average amount of light emission from the test substancenon-supplemented wells (n=5)

The antibody-drug conjugates (8) and (9) exhibited a cytotoxic activityof IC₅₀<0.1 (nM) against the HL-cells. On the other hand, theantibody-drug conjugates (8) and (9) exhibited no cytotoxic activityagainst the Raji cells (>100 (nM)). The anti-CD33 antibody exhibited nocytotoxic activity against both of the cells (>100 (nM)).

(Test Example 7) Cytotoxicity Test (5) of Antibody-Drug Conjugate

Antigen-positive cells NOMO-1 cells (HSRRB) were cultured in RPMI1640(GIBCO) containing 10% fetal bovine serum (MOREGATE) (hereinafter,referred to as a medium). The HOMO-1 cells were prepared to have aconcentration of 2.8×10⁴ cells/mL by using a medium and added at aconcentration of 90 μL/well to a 96-well microplate for cell culture.Two hours later, the anti-CD33 antibody and antibody-drug conjugates(24), (25), and (67) each diluted into 1000 nM, 200 nM, 40 nM, 8 nM, 1.6nM, 0.32 nM, and 0.064 nM by using a medium were added at aconcentration of 10 μL/well to the microplate. A medium was added at aconcentration of 10 μL/well to test substance non-supplemented wells.The cells were cultured under 5% CO₂ at 37° C. for 6 days. After theculture, the microplate was taken out from the incubator and leftstanding for 30 minutes at room temperature. The culture solution wascharged with an equal amount of CellTiter-Glo Luminescent Cell ViabilityAssay (Promega) and stirred. After the microplate was left standing atroom temperature for 10 minutes, the amount of light emission wasmeasured by using a plate reader (PerkinElmer). The IC₅₀ value wascalculated according to the following equation:

IC₅₀ (nM)=antilog((50−d)×(LOG₁₀ b−LOG₁₀ a)/(d−c)+LOG₁₀ b)

a: Concentration a of the test substance

b: Concentration b of the test substance

c: Ratio of live cells supplemented with the test substance having theconcentration a

d: Ratio of live cells supplemented with the test substance having theconcentration b

The concentrations a and b establish the relation a>b crossing 50% ratioof live cells.

The survival rate of cells at each concentration was calculatedaccording to the following equation:

Survival rate of cells (%)=a/b×100

a: Average amount of light emission from the test substance-supplementedwells (n=2)

b: Average amount of light emission from the test substancenon-supplemented wells (n=5)

The antibody-drug conjugate (25) exhibited a cytotoxic activity ofIC₅₀<0.1 (nM) against the NOMO-cells. The antibody-drug conjugates (24)and (67) exhibited a cytotoxic activity of 1<IC₅₀<100 (nM) against thecells. The anti-CD33 antibody exhibited no cytotoxic activity againstthe NOMO-1 cells (>100 (nM)).

(Test Example 8) Cytotoxicity Test (6) of Antibody-Drug Conjugate

Antigen-positive cells U251 cells (ATCC) or antigen-negative cells MCF-7cells (ATCC) were cultured in RPMI1640 (GIBCO) containing 10% fetalbovine serum (MOREGATE) (hereinafter, referred to as a medium). The U251cells or MCF-7 cells were prepared to have a concentration of 2.8×10⁴cells/mL by using a medium, added at a concentration of 90 μL/well to a96-well microplate for cell culture, and cultured overnight. On the nextday, the anti-CD70 antibody and antibody-drug conjugates (10) and (11)each diluted into 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, and0.064 nM by using a medium were added at a concentration of 10 μL/wellto the microplate. A medium was added at a concentration of 10 μL/wellto test substance non-supplemented wells. The cells were cultured under5% CO₂ at 37° C. for 6 days. After the culture, the microplate was takenout from the incubator and left standing at room temperature for 30minutes. The culture solution was charged with an equal amount ofCellTiter-Glo Luminescent Cell Viability Assay (Promega) and stirred.After the microplate was left standing at room temperature for 10minutes, the amount of light emission was measured by using a platereader (PerkinElmer). The IC₅₀ value was calculated according to thefollowing equation:

IC₅₀ (nM)=antilog((50−d)×(LOG₁₀ b−LOG₁₀ a)/(d−c)+LOG₁₀ b)

a: Concentration a of the test substance

b: Concentration b of the test substance

c: Ratio of live cells supplemented with the test substance having theconcentration a

d: Ratio of live cells supplemented with the test substance having theconcentration b

The concentrations a and b establish the relation a>b crossing 50% ratioof live cells.

The survival rate of cells at each concentration was calculatedaccording to the following equation:

Survival rate of cells (%)=a/b×100

a: Average amount of light emission from the test substance-supplementedwells (n=2)

b: Average amount of light emission from the test substancenon-supplemented wells (n=12)

The antibody-drug conjugates (10) and (11) exhibited a cytotoxicactivity of IC₅₀<1 (nM) against the U251 cells. On the other hand, theantibody-drug conjugates (10) and (11) exhibited no cytotoxic activityagainst the MCF-7 cells 90 (nM)). The anti-CD70 antibody exhibited nocytotoxic activity against both of the cells (>100 (nM)).

(Test Example 9) Cytotoxicity Test (7) of Antibody-Drug Conjugate

Antigen-positive cells U251 cells (ATCC) were cultured in RPMI1640(GIBCO) containing 10% fetal bovine serum (MOREGATE) (hereinafter,referred to as a medium). The U251 cells were prepared to have aconcentration of 2.8×10⁴ cells/mL by using a medium and added at aconcentration of 90 μL/well to a 96-well microplate for cell culture.Two hours later, the anti-CD70 antibody and antibody-drug conjugates(26), (27), (40), (68), and (69) each diluted into 1000 nM, 200 nM, 40nM, 8 nM, 1.6 nM, 0.32 nM, and 0.064 nM by using a medium were added ata concentration of 10 μL/well to the microplate. A medium was added at aconcentration of 10 μL/well to test substance non-supplemented wells.The cells were cultured under 5% CO₂ at 37° C. for 6 days. After theculture, the microplate was taken out from the incubator and leftstanding at room temperature for 30 minutes. The culture solution wascharged with an equal amount of CellTiter-Glo Luminescent Cell ViabilityAssay (Promega) and stirred. After the microplate was left standing atroom temperature for 10 minutes, the amount of light emission wasmeasured by using a plate reader (PerkinElmer). The IC₅₀ value wascalculated according to the following equation:

IC₅₀ (nM)=antilog((50−d)×(LOG₁₀ b−LOG₁₀ a)/(d−c)+LOG₁₀ b)

a: Concentration a of the test substance

b: Concentration b of the test substance

c: Ratio of live cells supplemented with the test substance having theconcentration a

d: Ratio of live cells supplemented with the test substance having theconcentration b

The concentrations a and b establish the relation a>b crossing 50% ratioof live cells.

The survival rate of cells at each concentration was calculatedaccording to the following equation:

Survival rate of cells (%)=a/b×100

a: Average amount of light emission from the test substance-supplementedwells (n=2)

b: Average amount of light emission from the test substancenon-supplemented wells (n=12)

The antibody-drug conjugates (26), (27), (40), and (69) exhibited acytotoxic activity of 1<IC₅₀<10 (nM) against the U251 cells. Theantibody-drug conjugate (68) exhibited a cytotoxic activity of10<IC₅₀<100 (nM) against the cells. The anti-CD70 antibody exhibited nocytotoxic activity against the U251 cells (>100 (nM)).

(Test Example 10) Cytotoxicity Test (8) of Released Drug

A375 cells (ATCC) were cultured in DMEM (GIBCO) containing 10% fetalbovine serum (MOREGATE) (hereinafter, referred to as a medium). The A375cells were prepared to have a concentration of 4×10⁴ cells/mL by using amedium, added at a concentration of 25 μL/well to a 96-well microplatefor cell culture (CORNING) charged with 65 μL/well of a medium, andcultured overnight. On the next day, each test substance diluted into1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, and 0.064 nM by usingDMSO was added at a concentration of 0.5 μL/well to the microplate. DMSOwas added at a concentration of 0.5 μL/well to test substancenon-supplemented wells. The volume of the medium in each well wasadjusted to 100 μL by the addition of 10 μL/well of a medium, and thecells were cultured under 5% CO₂ at 37° C. for 6 days. After theculture, the microplate was taken out from the incubator and leftstanding at room temperature for 30 minutes. The culture solution wascharged with an equal amount of CellTiter-Glo Luminescent Cell ViabilityAssay (Promega) and stirred. After the microplate was left standing atroom temperature for 10 minutes, the amount of light emission wasmeasured by using a plate reader. The IC₅₀ value was calculatedaccording to the following equation:

IC₅₀ (nM)=antilog((50−d)×(LOG₁₀ b−LOG₁₀ a)/(d−c)+LOG₁₀ b)

a: Concentration a of the test substance

b: Concentration b of the test substance

c: Ratio of live cells supplemented with the test substance having theconcentration a

d: Ratio of live cells supplemented with the test substance having theconcentration b

The concentrations a and b establish the relation a>b crossing 50% ratioof live cells.

The survival rate of cells was calculated according to the followingequation:

Survival rate of cells (%)=a/b×100

a: Average amount of light emission from the test substance-supplementedwells (n=2)

b: Average amount of light emission from the test substancenon-supplemented wells (n=10)

The compound of Example (75) and exatecan exhibited a cytotoxic activityof 0.1<IC₅₀<1 (nM) against the A375 cells. The compound of Example (42)exhibited a cytotoxic activity against 1<IC₅₀<10 (nM) against the cells.The compound of Example (1) exhibited a cytotoxic activity against10<IC₅₀<100 (nM) against the cells.

(Test Example 11) Antitumor test (1)

Mouse: 5- to 6-week-old female BALB/c nude mice (Charles RiverLaboratories Japan, Inc.) were acclimatized for 4 to 7 days under SPFconditions before use in the experiment. The mice were fed withsterilized solid feed (FR-2, Funabashi Farms Co., Ltd) and givensterilized tap water (prepared by the addition of 5 to 15 ppm sodiumhypochlorite solution).

Assay and calculation expression: In all studies, the major axis andminor axis of tumor were measured twice a week by using an electronicdigital caliper (CD-15C, Mitutoyo Corp.), and the tumor volume (mm³) wascalculated. The calculation expression is as shown below.

Tumor volume (mm³)=½×Major axis (mm)×[Minor axis (mm)]²

All of the antibody-drug conjugates were diluted with physiologicalsaline (Otsuka Pharmaceutical Factory, Inc.) and used at a volume of 10mL/kg for intravenous administration to the tail of each mouse. Humanmelanoma line A375 cells were purchased from ATCC (American Type CultureCollection). 8×10⁶ cells suspended in physiological saline weresubcutaneously transplanted to the right abdomen of each female nudemouse (Day 0), and the mice were randomly grouped at Day 11. TheM30-H1-L4P antibody and antibody-drug conjugate (2) were eachintravenously administered at a dose of 10 mg/kg to the tail of eachmouse at Days 11, 18, and 25.

The results are shown in FIG. 17. In the drawing, the line with openrhombuses depicts the results about untreated tumor, the line with opentriangles depicts the effect of the M30-H1-L4P antibody, and the linewith open circles depicts the effect of the antibody-drug conjugate (2).

As seen from these results, the administration of the antibody-drugconjugate (2) remarkably decreased the tumor volume, and no furthertumor growth was observed after the final administration. By contrast,the administration of the M30-H1-L4P antibody resulted in theprogression of tumor growth.

In addition, the mice that received the antibody-drug conjugate (2) werefree from notable signs such as weight loss, suggesting that theantibody-drug conjugate (2) is low toxic and highly safe.

(Test Example 12) Antitumor test (2)

Human melanoma line A375 cells were purchased from ATCC (American TypeCulture Collection). 6×10⁶ cells suspended in physiological saline weresubcutaneously transplanted to the right abdomen of each female nudemouse (Day 0), and the mice were randomly grouped at Day 18. Theantibody-drug conjugate (2) was intravenously administered at each dose(0.1, 0.3, and 1.3 mg/kg) to the tail of each mouse at Days 18, 25, and32 in a schedule of qw×3.

The results are shown in FIG. 18. In the drawing, the line with openrhombuses depicts the results about untreated tumor, the line withfilled squares depicts the effect of the antibody-drug conjugate (2)administered at 0.1 mg/kg, the line with X marks depicts the effect ofthe antibody-drug conjugate (2) administered at 0.3 mg/kg, the line withfilled triangles depicts the effect of the antibody-drug conjugate (2)administered at 1 mg/kg, and the line with open circles depicts theeffect of the antibody-drug conjugate (2) administered at 3 mg/kg. Theantibody-drug conjugate (2) was effective for shrinking tumor in adose-dependent manner.

(Test Example 13) Antitumor Test (3)

Human non-small cell lung cancer line Calu-6 cells were purchased fromATCC (American Type Culture Collection). 5×10⁶ cells suspended inphysiological saline were subcutaneously transplanted to the rightabdomen of each female nude mouse (Day 0), and the mice were randomlygrouped at Day 11. The M30-H1-L4P antibody and antibody-drug conjugate(2) were each intravenously administered at a dose of 10 mg/kg to thetail of each mouse at Days 11, 18, and 25 in a schedule of qw×3.

The results are shown in FIG. 19. In the drawing, the line with openrhombuses depicts the results about untreated tumor, the line with opentriangles depicts the effect of the M30-H1-L4P antibody, and the linewith open circles depicts the effect of the antibody-drug conjugate (2).The administration of the antibody-drug conjugate (2) remarkablydecreased the tumor volume, and no further tumor growth was observedafter the final administration. By contrast, the administration of theM30-H1-L4P antibody resulted in the progression of tumor growth.

In addition, the mice that received the antibody-drug conjugate (2) werefree from notable signs such as weight loss, suggesting that theantibody-drug conjugate (2) is low toxic and highly safe.

(Test Example 14) Antitumor Test (4)

Human melanoma line A375 cells were purchased from ATCC (American TypeCulture Collection). 8×10⁶ cells suspended in physiological saline weresubcutaneously transplanted to the right abdomen of each female nudemouse (Day 0), and the mice were randomly grouped at Day 21. Theantibody-drug conjugates (1), (13), (41), and (55) were eachintravenously administered at a dose of 10 mg/kg to the tail of eachmouse at Day 21.

The results are shown in FIG. 20. In the drawing, the line with openrhombuses depicts the results about untreated tumor, the line with opencircles depicts the effect of the administered antibody-drug conjugate(1), the line with open triangles depicts the effect of the administeredantibody-drug conjugate (13), the line with X marks depicts the effectof the administered antibody-drug conjugate (41), and the line with opensquares depicts the effect of the administered antibody-drug conjugate(55). The administration of the antibody-drug conjugate (1), (13), (41),or (55) remarkably decreased the tumor volume, and all of theseantibody-drug conjugates exerted a tumor growth inhibitory effect.

In addition, the mice that received the antibody-drug conjugate (1),(13), (41), or (55) were free from notable signs such as weight loss,suggesting that the antibody-drug conjugates (1), (13), (41), and (55)are low toxic and highly safe.

(Test Example 15) Antitumor test (5)

Human non-small cell lung cancer line Calu-6 cells were purchased fromATCC (American Type Culture Collection). 5×10⁶ cells suspended inphysiological saline were subcutaneously transplanted to the rightabdomen of each female nude mouse (Day 0), and the mice were randomlygrouped at Day 12. The antibody-drug conjugates (13), (41), and (55)were each intravenously administered at a dose of 10 mg/kg to the tailof each mouse at Day 12. As a comparative control, DE-310 wasintravenously administered at a dose of 0.1 mg/kg to the tail of eachmouse at Day 12. Here, the aforementioned dose of the antibody-drugconjugate was based on the amount of the antibody in the conjugate andthe aforementioned dose of DE-310 was based on the amount of the drugcontained therein. In this respect, the amounts of the drugsrespectively contained in the antibody-drug conjugate and DE-310 wereabout 1:100. This means that the doses of the antibody-drug conjugateand DE-310 were equal in terms of the amounts of the drugs containedtherein.

The results are shown in FIG. 21. In the drawing, the line with openrhombuses depicts the results about untreated tumor, the line with opencircles depicts the effect of DE-310, the line with open trianglesdepicts the effect of the antibody-drug conjugate (13), the line with Xmarks depicts the effect of the antibody-drug conjugate (41), and theline with open squares depicts the effect of the antibody-drug conjugate(55). The administration of the antibody-drug conjugate (13), (41), or(55) remarkably decreased the tumor volume, whereas the administrationof DE-310 exhibited no reduction in tumor volume.

In addition, the mice that received the antibody-drug conjugate (13),(41), or (55) were free from notable signs such as weight loss,suggesting that these antibody-drug conjugates are low toxic and highlysafe.

(Test Example 16) Antitumor test (6)

Human melanoma line A375 cells were purchased from ATCC (American TypeCulture Collection). 1×10⁷ cells suspended in physiological saline weresubcutaneously transplanted to the right abdomen of each female nudemouse (Day 0), and the mice were randomly grouped at Day 11. Theantibody-drug conjugates (17), (18), (19), (59), (60), and (61) wereeach intravenously administered at a dose of 3 mg/kg to the tail of eachmouse at Days 11 and 18 in a schedule of qw×2.

The results are shown in FIG. 22. In the drawing, the line with filledrhombuses depicts the results about untreated tumor, the line withfilled squares depicts the effect of the administered antibody-drugconjugate (17), the line with open triangles depicts the effect of theadministered antibody-drug conjugate (18), the line with open circlesdepicts the effect of the administered antibody-drug conjugate (19), theline with filled triangles depicts the effect of the administeredantibody-drug conjugate (59), the line with open squares depicts theeffect of the administered antibody-drug conjugate (60), and the linewith X marks depicts the effect of the administered antibody-drugconjugate (61).

The administration of the antibody-drug conjugate (17), (18), (19),(59), (60), or (61) remarkably decreased the tumor volume, and all ofthese antibody-drug conjugates exerted a tumor growth inhibitory effect.

In addition, the mice that received the antibody-drug conjugate (17),(18), (19), (59), (60), or (61) were free from notable signs such asweight loss, suggesting that these antibody-drug conjugates are lowtoxic and highly safe.

Free Text of Sequence Listing

SEQ ID NO: 1—Amino acid sequence of the B7-H3 variant 1SEQ ID NO: 2—Amino acid sequence of the B7-H3 variant 2SEQ ID NO: 3—Amino acid sequence of CDRH1 of the M30 antibodySEQ ID NO: 4—Amino acid sequence of CDRH2 of the M30 antibodySEQ ID NO: 5—Amino acid sequence of CDRH3 of the M30 antibodySEQ ID NO: 6—Amino acid sequence of CDRL1 of the M30 antibodySEQ ID NO: 7—Amino acid sequence of CDRL2 of the M30 antibodySEQ ID NO: 8—Amino acid sequence of CDRL3 of the M30 antibodySEQ ID NO: 9—Amino acid sequence of the M30-H1-type heavy chainSEQ ID NO: 10—Amino acid sequence of the M30-H2-type heavy chainSEQ ID NO: 11—Amino acid sequence of the M30-H3-type heavy chainSEQ ID NO: 12—Amino acid sequence of the M30-H4-type heavy chainSEQ ID NO: 13—Amino acid sequence of the M30-L1-type light chainSEQ ID NO: 14—Amino acid sequence of the M30-L2-type light chainSEQ ID NO: 15—Amino acid sequence of the M30-L3-type light chainSEQ ID NO: 16—Amino acid sequence of the M30-L4-type light chainSEQ ID NO: 17—Amino acid sequence of the M30-L5-type light chainSEQ ID NO: 18—Amino acid sequence of the M30-L6-type light chainSEQ ID NO: 19—Amino acid sequence of the M30-L7-type light chainSEQ ID NO: 20—Amino acid sequence of a heavy chain of the M30 antibodySEQ ID NO: 21—Amino acid sequence of a light chain of the M30 antibodySEQ ID NO: 22—PCR primer 1SEQ ID NO: 23—PCR primer 2SEQ ID NO: 24—CMV promoter primer: primer 3SEQ ID NO: 25—BGH reverse primer: primer 4SEQ ID NO: 26—Nucleotide sequence of the B7-H3 variant 1SEQ ID NO: 27—Amino acid sequence of a heavy chain of the anti-CD30antibodySEQ ID NO: 28—Amino acid sequence of a light chain of the anti-CD30antibodySEQ ID NO: 29—Amino acid sequence of a heavy chain of the anti-CD33antibodySEQ ID NO: 30—Amino acid sequence of a light chain of the anti-CD33antibodySEQ ID NO: 31—Amino acid sequence of a heavy chain of the anti-CD70antibodySEQ ID NO: 32—Amino acid sequence of a light chain of the anti-CD70antibody

1. An antibody-drug conjugate, wherein a linker and an antitumorcompound represented by the following formula and anti-B7-H3 antibodyare connected:-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)wherein -(Succinimid-3-yl-N)— has a structure represented by thefollowing formula:

which is connected to the antibody at position 3 thereof and isconnected to a methylene group in the linker structure containing thisstructure on the nitrogen atom at position 1, and (NH-DX) represents agroup represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is theconnecting position, the anti-B7-H3 antibody has a CDRH1 comprising anamino acid sequence represented by SEQ ID NO: 3, a CDRH2 comprising anamino acid sequence represented by SEQ ID NO: 4, and a CDRH3 comprisingan amino acid sequence represented by SEQ ID NO: 5 as heavy chaincomplementarity determining regions, and a CDRL1 comprising an aminoacid sequence represented by SEQ ID NO: 6, a CDRL2 comprising an aminoacid sequence represented by SEQ ID NO: 7, and a CDRL3 comprising anamino acid sequence represented by SEQ ID NO: 8 as light chaincomplementarity determining regions.
 2. The antibody-drug conjugateaccording to claim 1, wherein the anti-B7-H3 antibody has a heavy chainvariable region and a light chain variable region selected from thegroup consisting of a heavy chain variable region comprising an aminoacid sequence described in amino acid positions 20 to 141 in SEQ ID NO:9 and a light chain variable region comprising an amino acid sequencedescribed in amino acid positions 21 to 128 in SEQ ID NO: 13, a heavychain variable region comprising an amino acid sequence described inamino acid positions 20 to 141 in SEQ ID NO: 9 and a light chainvariable region comprising an amino acid sequence described in aminoacid positions 21 to 128 in SEQ ID NO: 14, a heavy chain variable regioncomprising an amino acid sequence described in amino acid positions 20to 141 in SEQ ID NO: 9 and a light chain variable region comprising anamino acid sequence described in amino acid positions 21 to 128 in SEQID NO: 15, a heavy chain variable region comprising an amino acidsequence described in amino acid positions 20 to 141 in SEQ ID NO: 9 anda light chain variable region comprising an amino acid sequencedescribed in amino acid positions 21 to 128 in SEQ ID NO: 16, a heavychain variable region comprising an amino acid sequence described inamino acid positions 20 to 141 in SEQ ID NO: 9 and a light chainvariable region comprising an amino acid sequence described in aminoacid positions 21 to 128 in SEQ ID NO: 17, a heavy chain variable regioncomprising an amino acid sequence described in amino acid positions 20to 141 in SEQ ID NO: 9 and a light chain variable region comprising anamino acid sequence described in amino acid positions 21 to 128 in SEQID NO: 18, a heavy chain variable region comprising an amino acidsequence described in amino acid positions 20 to 141 in SEQ ID NO: 9 anda light chain variable region comprising an amino acid sequencedescribed in amino acid positions 21 to 128 in SEQ ID NO: 19, a heavychain variable region comprising an amino acid sequence described inamino acid positions 20 to 141 in SEQ ID NO: 12 and a light chainvariable region comprising an amino acid sequence described in aminoacid positions 21 to 128 in SEQ ID NO: 13, a heavy chain variable regioncomprising an amino acid sequence described in amino acid positions 20to 141 in SEQ ID NO: 12 and a light chain variable region comprising anamino acid sequence described in amino acid positions 21 to 128 in SEQID NO: 14, a heavy chain variable region comprising an amino acidsequence described in amino acid positions 20 to 141 in SEQ ID NO: 12and a light chain variable region comprising an amino acid sequencedescribed in amino acid positions 21 to 128 in SEQ ID NO: 15, and aheavy chain variable region comprising an amino acid sequence describedin amino acid positions 20 to 141 in SEQ ID NO: 12 and a light chainvariable region comprising an amino acid sequence described in aminoacid positions 21 to 128 in SEQ ID NO:
 16. 3. The antibody-drugconjugate according to claim 1, wherein the anti-B7-H3 antibodycomprises a heavy chain and a light chain selected from the groupconsisting of a heavy chain comprising an amino acid sequence describedin amino acid positions 20 to 471 in SEQ ID NO: 9 and a light chaincomprising an amino acid sequence described in amino acid positions 21to 233 in SEQ ID NO: 13, a heavy chain comprising an amino acid sequencedescribed in amino acid positions 20 to 471 in SEQ ID NO: 9 and a lightchain comprising an amino acid sequence described in amino acidpositions 21 to 233 in SEQ ID NO: 14, a heavy chain comprising an aminoacid sequence described in amino acid positions 20 to 471 in SEQ ID NO:9 and a light chain comprising an amino acid sequence described in aminoacid positions 21 to 233 in SEQ ID NO: 15, a heavy chain comprising anamino acid sequence described in amino acid positions 20 to 471 in SEQID NO: 9 and a light chain comprising an amino acid sequence describedin amino acid positions 21 to 233 in SEQ ID NO: 16, a heavy chaincomprising an amino acid sequence described in amino acid positions 20to 471 in SEQ ID NO: 9 and a light chain comprising an amino acidsequence described in amino acid positions 21 to 233 in SEQ ID NO: 17, aheavy chain comprising an amino acid sequence described in amino acidpositions 20 to 471 in SEQ ID NO: 9 and a light chain comprising anamino acid sequence described in amino acid positions 21 to 233 in SEQID NO: 18, a heavy chain comprising an amino acid sequence described inamino acid positions 20 to 471 in SEQ ID NO: 9 and a light chaincomprising an amino acid sequence described in amino acid positions 21to 233 in SEQ ID NO: 19, a heavy chain comprising an amino acid sequencedescribed in amino acid positions 20 to 471 in SEQ ID NO: 12 and a lightchain comprising an amino acid sequence described in amino acidpositions 21 to 233 in SEQ ID NO: 13, a heavy chain comprising an aminoacid sequence described in amino acid positions 20 to 471 in SEQ ID NO:12 and a light chain comprising an amino acid sequence described inamino acid positions 21 to 233 in SEQ ID NO: 14, a heavy chaincomprising an amino acid sequence described in amino acid positions 20to 471 in SEQ ID NO: 12 and a light chain comprising an amino acidsequence described in amino acid positions 21 to 233 in SEQ ID NO: 15,and a heavy chain comprising an amino acid sequence described in aminoacid positions 20 to 471 in SEQ ID NO: 12 and a light chain comprisingan amino acid sequence described in amino acid positions 21 to 233 inSEQ ID NO:
 16. 4. The antibody-drug conjugate according to claim 3,wherein the anti-B7-H3 antibody lacks an amino acid at the carboxylterminus of the amino acid sequence represented by SEQ ID NO: 9 or 12 inthe heavy chain.
 5. The antibody-drug conjugate according to claim 1,wherein the anti-B7-H3 antibody has a heavy chain variable regioncomprising an amino acid sequence described in amino acid positions 20to 141 in SEQ ID NO: 9 and a light chain variable region comprising anamino acid sequence described in amino acid positions 21 to 128 in SEQID NO:
 16. 6. The antibody-drug conjugate according to claim 1, whereinthe anti-B7-H3 antibody comprises a heavy chain comprising an amino acidsequence described in amino acid positions 20 to 471 in SEQ ID NO: 9 anda light chain comprising an amino acid sequence described in amino acidpositions 21 to 233 in SEQ ID NO:
 16. 7. The antibody-drug conjugateaccording to claim 6, wherein the anti-B7-H3 antibody lacks an aminoacid at the carboxyl terminus of the amino acid sequence represented bySEQ ID NO: 9 in the heavy chain.
 8. The antibody-drug conjugateaccording to claim 1, wherein an average number of units of thedrug-linker structure conjugated per antibody is in a range of from 2 to8.
 9. The antibody-drug conjugate according to claim 1, wherein anaverage number of units of the drug-linker structure conjugated perantibody is in a range of from 3 to
 8. 10. A drug containing theantibody-drug conjugate according to claim 1 or a salt thereof.
 11. Anantitumor drug and/or anticancer drug containing the antibody-drugconjugate according to claim 1 or a salt thereof.
 12. A method oftreating cancer in an individual comprising administering to anindividual with cancer the drug according to claim 11, wherein thecancer is lung cancer, kidney cancer, urothelial cancer, colon cancer,prostate cancer, glioblastoma multiforme, ovarian cancer, pancreaticcancer, breast cancer, melanoma, liver cancer, bladder cancer, stomachcancer, or esophageal cancer.
 13. A pharmaceutical compositioncontaining the antibody-drug conjugate according to claim 1 or a saltthereof as an active component, and a pharmaceutically acceptableformulation component.
 14. A method of treating cancer in an individualcomprising administering to an individual with cancer the pharmaceuticalcomposition according to claim 13, wherein the cancer is lung cancer,kidney cancer, urothelial cancer, colon cancer, prostate cancer,glioblastoma multiforme, ovarian cancer, pancreatic cancer, breastcancer, melanoma, liver cancer, bladder cancer, stomach cancer, oresophageal cancer.